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SPR Biosensing in Crude Serum Using Ultralow Fouling Binary Patterned Peptide SAM
Abstract
Near-zero fouling monolayers based on binary patterned peptides allow low nanomolar detection of the matrix metalloproteinase-3 (MMP-3) directly in crude bovine serum, without sample pretreatment, secondary antibody detection or signal amplification. The peptide 3-MPA-HHHDD-OH (3-MPA, 3-mercaptopropionic acid) was found optimal compared to other binary patterned peptides based on 3-MPA-A(x)-B(y)-OH, where 0 <or= x, y <or= 5, and x + y = 5, and compared to PEG. In this study, amino acid A was His, Asp, Ser, or Leu, and amino acid B was His, Asp, or Ser. Zwitterionic peptides and other peptides exhibited excellent resistance to nonspecific adsorption. Binary patterned peptides were capped with 3-MPA on the N-terminus providing a monolayer with the C-terminus carboxylic acid available to subsequently immobilize antibodies. Thereby, an IgG biosensor demonstrated the efficiency of binary patterned peptides in SPR biosensing with a detection limit of 1-10 pM in PBS, similar to other optical or electrochemical techniques. This protocol was applied to establish a calibration curve for MMP-3, an analyte of clinical interest for many pathologies and a potential indicator of cancer. The LOD for MMP-3 was 0.14 nM in PBS, with a linearity of up to 50 nM. With the use of PBS calibration, MMP-3 was quantified at low nanomolar in undiluted bovine serum. The SPR response in serum was statistically the same as in PBS. A sensor exposed to blank serum exhibited negligible nonspecific adsorption. Hence, binary patterned peptides are suitable for biosensing directly in complex biological matrixes.
... The latter (N LOD ) relies mostly on the intrinsic properties of the sensor, which are severely affected by the non-specific interactions especially for sensor operation in a complex clinical sample. However, we find that recent progress on biochemistry [108] has been made to develop reliable schemes for molecular immobilization yielding increased antibody surface coverage density. Proper surface biofunctionalization can also lead to enhanced sensitivity by reducing background noise due to non-specific interactions of the biomolecules. ...
... Proper surface biofunctionalization can also lead to enhanced sensitivity by reducing background noise due to non-specific interactions of the biomolecules. Together with this, novel nanofabrication and nanomaterial patterning techniques may produce nanosturctured surfaces with rough inhomogenous features [109], self-assembly monolayer [108], or, parallelly patterned nanosensors [85]. These nanostructured surfaces will further enhance the sensitivity of label-free biosensors based on any transduction modality with a high surface-to-volume ratio. ...
... The latter (N Lod ) relies mostly on the intrinsic properties of the sensor, which are severely affected by the non-specific interactions especially for sensor operation in a complex clinical sample. However, we find that recent progress on biochemistry [108] has been made to develop reliable schemes for molecular immobilization yielding increased antibody surface coverage density. Proper surface biofunctionalization can also lead to enhanced sensitivity by reducing background noise due to non-specific interactions of the biomolecules. ...
... Proper surface biofunctionalization can also lead to enhanced sensitivity by reducing background noise due to non-specific interactions of the biomolecules. Together with this, novel nanofabrication and nanomaterial patterning techniques may produce nanosturctured surfaces with rough inhomogenous features [109], self-assembly monolayer [108], or, parallelly patterned nanosensors [85]. These nanostructured surfaces will further enhance the sensitivity of label-free biosensors based on any transduction modality with a high surface-to-volume ratio. ...
... In general, anti-fouling agents are added to surfaces in an attempt to tune the fouling properties of the surface (Fig. 1). Many different strategies have been explored for the prevention of NSA on surfaces2021222324. Immobilization of amino acids (i.e. ...
... Immobilization of amino acids (i.e. cysteine, gluthathione) or small oligopeptides onto gold have shown up to an 80% reduction of NSA212223. In 2010, Jeyachandran et al. explored the anti-fouling properties of bovine serum albumin (BSA) on hydrophobic and hydrophilic surfaces. ...
Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab’) in biosensors. One Fab’ linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab’ fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab’ fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab’-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.
... Commercial SPR sensors are often coated with a dextran-based coating which greatly reduces NSB from single protein solutions or biofluids diluted ten times; however, significant fouling occurs (>800 ng cm −2 ) when exposed to undiluted serum. [96,100,101] Therefore, integration with a high-performing nonfouling surface is vital for detection from complex biological milieu. [102] Although not intended to be a comprehensive review of SPR biosensors, several examples are summarized in the following. ...
“Nonfouling” polymer brush surfaces can greatly improve the performance of in vitro diagnostic (IVD) assays due to the reduction of nonspecific protein adsorption and consequent improvement of signal‐to‐noise ratios. The development of synthetic polymer brush architectures that suppress adventitious protein adsorption is reviewed, and their integration into surface plasmon resonance and fluorescent sandwich immunoassay formats is discussed. Also, highlighted is a novel, self‐contained immunoassay platform (the D4 assay) that transforms time‐consuming laboratory‐based assays into a user‐friendly and point‐of‐care format with a sensitivity and specificity comparable or better than standard enzyme‐linked immunosorbent assay (ELISA) directly from unprocessed samples. These advancements clearly demonstrate the utility of nonfouling polymer brushes as a substrate for ultrasensitive and robust diagnostic assays that may be suitable for clinical testing, in field and laboratory settings. The development of nonfouling polymer brush surfaces has ushered in a new wave of in vitro diagnostic (IVD) tests. Polymer brushes eliminate nonspecific protein adsorption to the surface and thus enable ultrasensitive detection of proteins directly from complex biological milieu. Diagnostic platforms capable of point‐of‐care testing are highlighted.
... While it is usually possible to control the environmental conditions such as temperature, reducing the effect of unwanted binding events in clinical samples is far more challenging. Different methods of overcoming this critical issue have been proposed, all based on surface chemistry approaches, using for example NHS esters [14], CM dextran [15], or polyethylene glycol (PEG) [16,17]. Integrating PEGs with silica microsphere resonators has for example allowed thrombin (~8 µM) to be detected in tenfold diluted human serum inside a flow cell [11]. ...
Although whispering gallery mode (WGM) biosensors have shown tremendous potential, they are still yet to find practical use as biomedical diagnostic tools. This is primarily due to the nature of the interrogation mechanism itself which relies on indirect measurement of the binding of a specific biomolecule onto the sensor through the associated refractive index change. Since nonspecific binding cannot be differentiated from the specific interaction of interest, this can result in a high rate of false positive readings when the detection is performed in complex biological samples. Here we show that this inherent limitation can be solved using a relatively simple approach. This approach involves the development of a self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a suspended core optical fiber. Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted human serum samples. The fiber allows remote excitation and collection of the WGMs of the microspheres in a dip sensing setting. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events, while the other microsphere is functionalized to detect the specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.
... Additionally, peptides may be used concurrently with other recognition elements to function as excellent suppressors of nonspecific binding. Thiolated oligonucleotides are easy to incorporate as diluents in gold sensors [16,17], and the effect of various end terminals on antifouling capabilities has been explored [18]. ...
Electrochemical biosensing represents a collection of techniques that may be utilized for capture and detection of biomolecules in both simple and complex media. While the instrumentation and technological aspects play important roles in detection capabilities, the interfacial design aspects are of equal importance, and often, those inspired by nature produce the best results. This review highlights recent material designs, recognition schemes, and method developments as they relate to targeted electrochemical analysis for biological systems. This includes the design of electrodes functionalized with peptides, proteins, nucleic acids, and lipid membranes, along with nanoparticle mediated signal amplification mechanisms. The topic of hyphenated surface plasmon resonance assays is also discussed, as this technique may be performed concurrently with complementary and/or confirmatory measurements. Together, smart materials and experimental designs will continue to pave the way for complete biomolecular analyses of complex and technically challenging systems.
... SPR and LSPR sensors can detect large biomolecules directly and with high sensitivity, such that it has become a widely popular technique for sensing disease markers. The recent advent of SPR techniques working in crude biofluids such as serum [13,14] and whole blood [15] makes these plasmonic sensors ideally suited for point-of-care applications. A broad range of sensors have been developed utilizing the variants on SPR on a thin metallic film [10], on nanoparticles [11] and on nanostructured metallic surfaces [16] for the detection of proteins, DNA, enzymes, drugs, and several other molecules and as such is an excellent example of a surface spectroscopy amenable to the detection of disease-related biomarkers. ...
This chapter talks about nanohole arrays that are especially interesting for the combination of plasmonic techniques alongside electrochemical measurements, as they are simultaneously electrically conductive and support localized and propagating plasmons necessary for surface-enhanced techniques. It presents a brief introduction to the mechanisms of surface-enhanced Raman scattering (SERS) enhancement resulting from plasmonic nanostructures. Electrochemical-SERS (EC-SERS) not only provides a platform for label-free detection, with the ability to easily prepare nanostructured electrodes and the miniaturization of SERS instruments, but also potentially provides a simple, rapid, and portable method of analysis ideally placed to be used in future instances of disease diagnostics and management. The most common biomolecules targeted in the field of disease diagnostics include DNA, which codes for genes specific to a certain disease state, as well as antigens indicative of the presence of a disease.
... 106,107 Masson and co-workers have used peptide-based coatings to minimize the nonspecific absorption of biological media. 108,109 Zwitterionic monolayers have also been widely studied as an efficient way to prevent nonspecific adhesion and demonstrated ultralow fouling properties. 105,110,111 3.3.2. ...
In an ideal plasmonic surface sensor, the bioactive area, where analytes are recognized by specific biomolecules, is surrounded by an area that is generally composed of a different material. The latter, often the surface of the supporting chip, is generally hard to be selectively functionalized with respect to the active area. As results, cross talks between the active area and the surrounding one may occur. In designing a plasmonic sensor, various issues must be addressed: the specificity of analyte recognition, the orientation of the immobilized biomolecule that acts as the analyte receptor and the selectivity of surface coverage. This tutorial review aims to introduce the main rational tools required for a correct and complete approach to chemically functionalize plasmonic surface biosensors. After a short introduction, the review discusses in detail the most common strategies for achieving effective surface functionalization. The most important issues, such as the orientation of active molecules and spatial and chemical selectivity, are considered. A list of well-defined protocols are suggested for the most common practical situations. Importantly, for the reported protocols, we also present direct comparisons in term of costs, labor demand and risk vs benefit balance. In addition, a survey of the most used characterization techniques necessary to validate the chemical protocols is reported.
... This has good prospects for the analysis and application of rapid biointeraction process. [36] Petide SAM 2 Human IgG 0.45 ng/mL (3 pM) <1 min [37] PSPW 3 Mouse IgG 10 pg/mL <1 s [15] Fluorescence Fluorescence microsope Horse IgG 0.71 µg/mL 25 min [38] Electrochemistry CAuNCs 4 Rabbit IgG 5 ng/mL <1 s [39] ELISA 5 Goat IgG 1 ng/mL 2-5 min [40] Ellipsometry RCE 6 + Porous silicon Albumin<10 s [41] Imaging AFP 7 5 ng/mL <1 s [22] SPRE SPR + Ellipsometry β-Cyclodextrins 1 pg/mL (1 pM) 2 s [25] Our platform 45 • dual-drive symmetric PEM + Bare Si Human IgG 15 ng/mL 1 ms ...
In this work, a biosensing method based on in situ, fast, and sensitive measurements of ellipsometric parameters (Ψ, ∆) is proposed. Bare silicon wafer substrate is functionalized and used to bind biomolecules in the solution. Coupled with a 45° dual-drive symmetric photoelastic modulator-based ellipsometry, the parameters Ψ and ∆ of biolayer arising due to biomolecular interactions are determined directly, and the refractive index (RI) of the solution and the effective thickness and surface mass density of the biolayer for various interaction time can be further monitored simultaneously. To illustrate the performance of the biosensing method, immunosensing for immunoglobulin G (IgG) was taken as a case study. The experiment results show that the biosensor response of the limit of detection for IgG is 15 ng/mL, and the data collection time is in milliseconds. Moreover, the method demonstrates a good specificity. Such technique is a promising candidate in developing a novel sensor which can realize fast and sensitive, label-free, easy operation, and cost-effective biosensing.
... This pCB architecture achieved ultralow protein fouling (<5 ng/cm 2 ) and high antibody loading ($1300 ng/cm 2 ). In peptide-based materials, Bolduc et al.[164]constructed a binary patterned peptide matrix to detect metalloproteinase-3 with an LOD of 0.14 nM. The response signals showed negligible changes when the matrix was used in crude bovine serum compared to that in phosphatebuffered saline, indicating similar adsorption levels. ...
Statement of significance:
Surface plasmon resonance (SPR) is a powerful tool in monitoring biomolecular interactions. The principle of SPR biosensors is the conversion of refractive index change caused by molecular binding into resonant spectral shifts. However, the fouling on the surface of SPR gold chips is ubiquitous and troublesome. It limits the application of SPR biosensors by blocking recognition element immobilization and specific binding. Hence, we write this paper to review the antifouling mechanisms and the recent advances of the design of antifouling materials that can improve the accuracy and sensitivity of SPR biosensors. To our knowledge, this is the first review focusing on the antifouling materials that were applied or had potential to be applied on SPR biosensors.
... For every biosensing analysis, a selfassembled monolayer (SAM) of 3-MPA-LHDLHD-OH peptide was formed on the sensors' surface as described in previous reports. 24 The plasmonic signal was measured 3 times for each well during a sequence. A total of 3 sequences was done for each measurements steps; thus, every sensor in the multiwell plate was scanned 9 times to measure repeatability. ...
A multi-well plasmonic reader was designed and validated for higher throughput analysis of biological interactions with a platform of the same size as standard 96-well plates. While the plasmonic sensor can be read with standard 96-well plate readers, a custom 96-well plate reader was designed to analyze nanohole arrays at high incident angles required for higher sensitivity. Gold nanohole arrays were manufactured on a 4 inch glass wafer using a photolithographic process. In comparison to single channel measurements with nanohole arrays fabricated with nanosphere lithography, the nanohole array sensors greatly enhanced the signal to noise ratio of the plasmonic signal and precision of the measurements with the multi-well plate system. As proof of concept, the detection of IgG in the low nanomolar range was achieved with the multi-well plate reader. The multi-well plasmonic plate reader was also applied to the screening of several prostate specific (PSA) antibodies for secondary detection of PSA and for the analysis of an anti-cancer drug through a competitive assay between methotrexate (MTX) and folic acid Au nanoparticle (FaNP) for human dihydrofolate reductase (hDHFR). The multi-well plasmonic reader based on nanohole array technology offers the rapid, versatile, sensitive and simple high throughput detection of biomolecules.
... These details, however, are limited: whereas structural studies of proteins in solution or in the crystal phase can be performed through techniques such as X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS), protein structures at interfaces remain elusive owing to low resolution and/ or a very small amount of available high resolution data (e.g., structural constraints). 13 −15 In fact, as McPherson et al. noted, 16 the difficulty in characterizing polymers near a surface has amounted to the neglect of many publications to note the surface density of grafted PEG chains. Despite the challenges associated with surface characterization, the field has developed substantial theory related to nonfouling. ...
Recent developments in the anti-fouling properties of Self-Assembled Monolayers (SAMs) have largely focused on increasing the enthalpic association of a hydration layer along the interface of those surfaces with water. However, an entropic penalty due to chain restriction also disfavors biomolecule-surface adsorption. To isolate the effect of this entropic penalty amid changing packing densities, molecular dynamics simulations of explicitly solvated systems of lysozyme and seven monomer length oligo (ethylene glycol) (OEG) SAMs were performed. SAM surfaces were constructed at 100%, 74%, and 53% of a maximum packing (MP) density of 4.97 Angstrom inter-chain spacing and the effect of chain flexibility was isolated by selectively freezing chain monomers. The rate of protein adsorption as well as the conformation and orientation of the protein upon adsorption were examined. It was found that chain spacing was a strong determinant in adsorption properties while chain flexibility played a secondary role. Of the three packing densities, 74% of MP was the most antifouling with increased antifouling behavior at moderate chain flexibility, i.e. two to four free monomer groups.
... La fonctionnalisation de l'or par un seul type d'alkanethiol a été développée dans de très nombreux travaux [ 204 ], mais l'utilisation de deux composés alkanethiol de nature et de propriétés différentes apporte un gain dans la biodétection [ 205,206 ] ainsi que dans la passivation des surfaces vis-à-vis des milieux biologiques complexes [207][208][209]. Le choix se portera alors sur une couche de biofonctionnalisation de thiol mixte: Le premier mécanisme est responsable de l'anneau observé après séchage d'une goutte de café par exemple et est appelé effet de bord ou effet tache de café ( Figure 61B). ...
The analytic approach of interrogation on chip by mass spectrometry is a suitable technique tomultiplexed analysis required for biomarker research in modern diagnosis. The aim was to contributeto the technological and methodological developments of analysis platform called SUPRA-MS(Surface Plasmon Resonance in Array coupled to Mass Spectrometry) whose goal is to provideadditional data to assay on the target protein by mass spectrometry. At first, gold chips compatiblewith SPRi and MS were designed and fabricated using vacuum deposition techniques. An originalstudy coupling SPRi with AFM has established a relationship between the SPR signal measured andthe real amount of proteins bound to nanostructured chips. We developed an immobilization procedurein array format (spots 16-96) by covalent monolayer of specific antibodies directed against the proteinLAG3, a biomarker of breast cancer for multiplex analysis of human biological samples (plasma).Human plasma containing 300 ng/mL of LAG3 is injected to the chip surface. The injection ismonitored in real time by SPRi and leads to the biomarker capture at the femtomole scale. After thebiosensing step, a collective treatment of spots by spray for in situ protein digestion and matrixdeposition in view of a MS analysis was developed by Dr. Patrick Ducoroy's team (CLIPP-CHUDijon). The MS and MS-MS analysis by MALDI-TOF was developed to analyze all spotsautomatically and determine their peptide mapping leading to 100% of the biomarker identification.This technology does not require the use of specific markers, is suitable to the biomarkerscharacterization and discrimination of protein variants.
... Amino acids as building blocks are in general available in large quantities in nature and the preparation of peptides by emerging recombinant expression systems provides environmentally friendly and sustainable access to large quantities of the material at solvent-free and mild preparation conditions. 12−14 Over the past decade, low to ultra-low NSA fouling peptide selfassembled monolayers (SAMs) have been designed on the basis of one or more of the following properties: structure, 15,16 amphiphilicity, 17−19 hydrophilicity, 15,17,20,21 or zwitterionic 16,19,20,22−24 character. Only a few cases that we are aware of have studied the ability of peptide SAMs to resist settlement of marine organisms, demonstrating that SAMs are excellent model systems to screen the antifouling properties of peptide sequences. ...
A series of ultra-low fouling peptide self-assembled monolayers (SAMs) was developed to demonstrate how the effects of subtle sequence alterations determine the properties of peptide terminated SAMs and settlement and adhesion of two model fouling organisms, the green alga Ulva linza (U. linza) and the diatom Navicula perminuta (N. perminuta), and adsorption of two different proteins, fibrinogen and lysozyme. Insertion of the bulky, nonnatural amino acid α-aminoisobutyric acid (Aib) was examined for how it affects the peptide surfaces and performance in the assays. By exchanging the serine (S) of the sequence (SGKGSSGSS) with alanine (A), we slightly altered the hydrophobicity and found a reducing fouling by N. perminuta. The inclusion of Aib residues resulted in surface structural changes of the peptides from a mixture of β-sheet/random coil to strictly random coil and a decrease in the overall packing density by about 17 to 21%. Notably, these changes had little effect on the ability of the surface to resist non-specific adsorption of fibrinogen and lysozyme and attachment of N. perminuta. The sequences containing Aib were 50 to 84% better than without Aib against the settlement of the zoospore of U. linza. Furthermore, the inclusion of Aib helped to create peptides that were 100% resistant against enzymatic degradation by trypsin, whereas the peptides without Aib were 95% degraded after 4 hours.
... Surface plasmon resonance (SPR) analysis is a novel bioanalytical tool to analyze the interaction between proteins, DNA, enzymes, and other biomolecules [19]. By combining SPR analysis and molecular docking, heparanase had been identified as a target of aspirin for tumor metastasis, angiogenesis, and growth in cancer [20]. ...
Background:
Current therapies for multiple myeloma (MM) are associated with toxicity and resistance, highlighting the need for novel effective therapeutics. Berberine (BBR), a botanical alkaloid derived from several Berberis medicinal plants, has exhibited anti-tumor effects, including against multiple myeloma (MM); however, the molecular mechanism underlying the anti-MM effect has not been previously described. This study aimed to identify the target of berberine and related mechanisms involved in its therapeutic activity against MM.
Results:
Here, we demonstrated that BBR treatment killed MM cells in vitro and prolonged the survival of mice bearing MM xenografts in vivo. A screening approach integrating surface plasmon resonance (SPR) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified UHRF1 (ubiquitin-like with PHD and RING Finger domains 1) as a potential target of BBR. Combining molecular docking and SPR analysis, we confirmed UHRF1 as a BBR-binding protein and discovered that BBR binds UHRF1 in the tandem tudor domain and plant homeodomain (TTD-PHD domain). BBR treatment induced UHRF1 degradation via the ubiquitin-dependent proteasome system and reactivated p16INK4A and p73 in MM cells. Overexpression of UHRF1 promoted the MM cell proliferation and rendered MM cells more resistant to BBR, while silencing of UHRF1 with siRNA attenuated BBR-induced cytotoxicity.
Conclusions:
In summary, our study has identified UHRF1 as a direct target of BBR and uncovered molecular mechanisms involved in the anti-MM activity of BBR. Targeting UHRF1 through BBR may be a novel therapeutic strategy against MM.
... As their biological functions are highly dependent on the proteolytic activity, the specific interrogation of the enzymatically active forms of MMPs is more clinically relevant (Lei et al., 2020). On the basis of the proteolytic cleavage of the recognition substrates, a series of methods have been presented for the interrogation of MMP activities, using such as gelatin zymography (Snoek-van Beurden and Von den Hoff, 2005), bioluminescence (Kim et al., 2010), porous silicon (Gupta et al., 2015), field effect transistors (FETs) (Chen et al., 2016), colorimetry (Chen et al., 2013;Gao et al., 2008;Kim et al., 2013), fluorescence (Gao et al., 2018;Li et al., 2011;Son et al., 2013), electrochemiluminescence (ECL) (Gao et al., 2017), surface-enhanced Raman scattering (SERS) (Gong et al., 2017), surface plasmon resonance (SPR) (Bolduc et al., 2010), and electrochemical sensors (Liu et al., 2006;Wang et al., 2016;Xi et al., 2020;Zheng et al., 2013;Zheng and Ma, 2018). In particular, considerable interest has been focused on the development of the cleavage-based electrochemical sensors, by virtue of their merits such as being low-cost, easy to fabricate, highly sensitive and selective, portable, and compatible with microfabrication. ...
Being closely associated with a variety of physiological and pathological processes, matrix metalloproteinases (MMPs) are useful as potential targets for drug therapy and informative markers for disease diagnosis. On the basis of the electrochemically induced grafting of ferrocenyl polymers and the proteolytic cleavage of recognition peptide, a novel electrochemical sensor is presented in this work for the highly specific interrogation of MMP activities at ultralow levels. The recognition peptide, to be immobilized via the N-terminus, is free of carboxyl group. The presence of the target MMP would cleave the end-tethered recognition peptide, generating a free carboxyl group at the C-terminus of the rest fragment. To be used as the reversible addition−fragmentation chain-transfer (RAFT) agent, the dithiobenzoate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD), can therefore be tethered via the carboxylate-Zr(IV)-carboxylate chemistry. Subsequently, the grafting of ferrocenyl polymers through electrochemically induced RAFT (eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA) would recruit a large quantity of Fc redox reporters on electrode surface. With benefits from the excellent specificity of the enzyme-substrate recognition, the presented cleavage-based sensor is highly selective. Under optimal conditions, the detection limit in the presence of MMP-2 as the model target can be as low as 0.27 pg mL⁻¹, with a linear range from 1 pg mL⁻¹ to 1 ng mL⁻¹. Furthermore, its applicability in the interrogation of MMP activity in complex serum samples and the screening of MMP inhibitors is satisfactory. The presented cleavage-based electrochemical MMP sensor is easy to fabricate and low-cost, thus showing great promise in drug discovery and disease diagnosis.
... The contact angles of such peptide SAMs were near the reported values of other antifouling peptides, such as an EKEKEKEPPPPC peptide SAM (20°) 36 and a 3-MPA-HHHDD−OH peptide SAM (35°). 39 The uniformity of the attached peptide SAMs on the gold substrates was assessed by AFM imaging. As shown in Figure 3, the AFM images showed that both peptide SAMs had a smooth surface with a characteristic root-mean-squared roughness of 0.8 nm, which was similar to that of the bare Au surface (0.7 nm), indicating uniform coverage of the Au surface. ...
The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption rates of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.
There are currently no biosensors that are able to reliably detect the process of cancer metastasis. We describe the first label-free real-time ultra-high frequency acoustic wave biosensor prototype capable of detecting the breast and prostate cancer metastasis biomarker, parathyroid hormone-related peptide (PTHrP). Two different linkers - 11-trichlorosilyl-undecanoic acid pentafluorophenyl ester (PFP) and S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) - were used to immobilize whole anti-PTHrP antibodies and Fab' fragments to surfaces as biorecognition elements. The biosensor surfaces were optimized using X-ray photoelectron spectroscopy (XPS) and the ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS). One optimized whole antibody-based surface (PFP/protein G'/whole antibodies/ethanolamine) and one optimized Fab' fragment-based surface (TUBTS/Fab' fragments) were tested as biosensors. It was determined that an in-line injection of bovine serum albumin prior to analyte injection yielded the most minimally fouling surfaces. Each surface was tested with no mass amplification and with sandwich-type secondary antibody mass amplification. The whole antibody-based mass-amplified biosensor yielded the lowest limit of detection (61ng/mL), highest sensitivity, and a linear range from 61ng/mL to 100μg/mL. However, the Fab' fragment-based biosensor displayed better regenerability as a loss of ~20% of the initial analyte signal intensity was observed with each subsequent injection. The whole antibody-based biosensor was only capable of producing an analyte signal in the first injection.
The present work reports on the synthesis of a redox-tagged peptide with self-assembling capability aiming applications in electrochemically active capacitive surfaces (associated with the presence of the redox centers) generally useful in electroanalytical applications. Peptide containing ferrocene (fc) molecular (redox) group (Ac-Cys-Ile-Ile-Lys(fc)-Ile-Ile-COOH) was thus synthesized by solid phase peptide synthesis (SPPS). To obtain the electrochemically active capacitive interface, the side chain of the cysteine was covalently bound to the gold electrode (sulfur group) and the side chain of Lys was used to attach the ferrocene in the peptide chain. After obtaining the purified redox-tagged peptide, the self-assembly and redox capability was characterized by cyclic voltammetry (CV) and electrochemical impedance-based capacitance spectroscopy techniques. The obtained results confirmed that the redox-tagged peptide was successfully attached by forming an electroactive self-assembled monolayer onto gold electrode. The design of redox active self-assembly ferrocene-tagged peptide is predictably useful in the development of biosensor devices precisely to detect, in a label-free platform, those biomarkers of clinical relevance. This article is protected by copyright. All rights reserved.
For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise towards meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of -concept study is carried out with 5 and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening.
Matrix metalloproteinases (MMPs) are believed to play an important role in tumor invasion. Herein, a peptide microarray-based fluorescence assay has been proposed for simultaneously determining the activities of MMP-2 and MMP-9 through the strong binding affinity of fluorescein isothiocyanate modified avidin (avidin-FITC) with the immobilized biotinylated peptide substrate on the microarray. In the presence of MMPs, the biotin moiety is released from the microarray by enzymatic cleavage of the peptide substrate, resulting in a significant decrease of the fluorescence signal. Under the optimal experimental conditions, the fluorescence intensity changes (ΔF%) are proportional to the concentrations of MMP-2 and MMP-9 within the ranges of 50 pg mL−1 to 50 ng mL−1 and 50 pg mL−1 to 100 ng mL−1 in the enzyme mixture, respectively. The detection limits are 45 pg mL−1 for MMP-2, and 60 pg mL−1 for MMP-9. In particular, the activities of extracellular MMP-2 and MMP-9 are determined by the peptide microarray-based fluorescence assay, and satisfactory results are obtained.
Biosensing within complex biological samples requires a sensor that can compensate for fluctuations in the signal due to changing environmental conditions and nonspecific binding events. To achieve this, we developed a novel self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a microstructured optical fiber (MOF). Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted, immunoglobulin-deprived human serum samples. The MOF allows remote excitation and collection of the whispering gallery modes (WGMs) of the microspheres while also providing a robust and easy to manipulate dip-sensing platform. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events and changes in the environment (such as refractive index and temperature), while the other microsphere is functionalized to detect a specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area, while still having discernible WGM spectra. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes, such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.
Nanobiosensors exploiting the optical phenomenon of surface plasmon resonance (SPR) are ideal candidates for the design of optical sensors for clinical diagnostics. The label-free nature and sensitivity of SPR biosensors to binding events makes them generally applicable to the detection of a broad class of biomolecules. Recent advances in instrument design, surface chemistry, nanomaterials and biosensing strategies have enabled numerous applications of SPR biosensors. This chapter will explore in greater detail the challenges and solutions developed recently for the analysis of proteins and drugs in crude biofluids. Specifically, surface chemistry has been investigated thoroughly to minimize the interference of nonspecific adsorption from biofluids, while nanomaterials have been exploited to increase the sensitivity of SPR biosensors, with biosensing strategies involving nanoparticles allowing for the analysis of small molecules. Additionally, miniaturization and optimization of instrumental design have paved the way towards point-of-care diagnostics. The successful detection of biologically relevant molecules directly in biofluids relies on all of these recent advances. In this chapter, they will be contextualized for the analysis of proteins and an oncologic drug, methotrexate, in crude serum samples using SPR sensing.
We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit anti-human IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit anti-human IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL-1 for MMP-2, and 5.7 pg mL-1 for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained.
Whispering gallery modes (WGMs) have been widely studied over the past 20 years for various applications, including biological sensing. While the WGM-based sensing approaches reported in the literature have shown tremendous performance down to single molecule detection, at present such sensing technologies are not yet mature and still have significant practical constraints that limit their use in real-world applications. Our work has focused on developing a practical, yet effective, WGM-based sensing platform capable of being used as a dip sensor for in-vivo biosensing by combining WGM fluorescent microresonators with silica Microstructured
Optical Fibers (MOFs).
We recently demonstrated that a suspended core MOF with a dye-doped polymer microresonator supporting WGMs positioned onto the tip of the fiber, can be used as a dip sensor. In this architecture the resonator is anchored to one of the MOF air holes, in contact with the fiber core, enabling a significant portion of the evanescent field from the fiber to overlap with the sphere and hence excite the fluorescent WGMs. This architecture allows for remote excitation and collection of the WGMs. The fiber also permits easy manipulation of the microresonator for dip sensing applications, and hence alleviates the need for a complex microfluidic interface. More importantly, it allows for an increase in both the excitation and collection efficiency compared to free space coupling, and also improves the Q factor.
In this paper we present our recent results on microstructured fiber tip WGM-based sensors and show that this sensing platform can be used in clinical diagnostics, for detecting various clinically relevant biomarkers in complex clinical samples.
Surface plasmon resonance (SPR) sensors have become valuable analytical sensors for biomolecule detection. While SPR is heralded with high sensitivity, label-free and real-time detection, nonspecific adsorption and detection of ultralow concentrations remain issues. Nonspecific adsorption can be minimized using adequate surface chemistry. For example, we have employed peptide monolayers to reduce nonspecific adsorption of crude serum or cell lysate. It is important to uncover the nature of molecules nonspecifically adsorbing to surfaces in these biofluids, to further improve understanding of the nonspecific adsorption processes. Mass spectrometry (MS) provides a complementary tool to SPR to identify biomolecule adsorbed to surface. Trypsic digestion of the proteins adsorbed to surfaces led to identification of characteristic peptides from the proteins involved in nonspecific adsorption. Nonspecific adsorption in crude cell lysate results mainly from lipids, as confirmed with SPR and MS but proteins were observed on some surfaces. In another application of SPR and MS, imaging SPR can be used in combination to imaging MS to image tissue sections. Thin sections of mouse liver were inserted in the fluidic chamber of a SPRi instrument and proteins were transferred to the SPRi chip. The SPR chip was then imaged using MALDI imaging MS to identify the biomolecules that were transferred to the SPRi chip.
Conventional cancer diagnostic techniques are not suitable for point-of-care testing because they are usually expensive, time consuming, labor intensive, and require large equipment. This paper presents a proof-of-concept biochip for point-of-care testing based on multiplex electrochemical detection. The microfluidic biochip is composed of inexpensive photoresist-polydimethylsiloxane layers over a glass substrate. The substrate has an Au working electrode and a Pt counter/reference electrode. A microchannel was fabricated in the photoresist layer and a micromixer was fabricated in the polydimethylsiloxane layer. Matrix metalloproteinases (MMPs) 2 and MMP7 are used as biomarkers for ovarian and colorectal cancer diagnosis. An unlabeled peptide specific to MMPs is immobilized on an Au electrode and then impedance was measured using electrochemical impedance spectroscopy with electrolytes. As the MMP sample is injected into the biochip, the peptide is cleaved by enzyme hydrolysis and the impedance changes. MMP2 and MMP7 are quantitatively detected concurrently by measuring the variations of impedance. Using model samples, the detection ranges of the biochip were 0.1-400 ng/mL and 0.001-100 ng/mL for MMP2 and MMP7, respectively, and the overall assay time was reduced to about 1 h. The results indicate that a similar MMP-based electrochemical multiplex biochip could be used for the point-of-care diagnosis of many forms of cancer.
We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures.
Recent progress in biointerface research has highlighted the role of antifouling functionalizable coatings in the development of advanced biosensors for point-of-care bioanalytical and biomedical applications dealing with real-world complex samples. The resistance to nonspecific adsorption promotes the biorecognition performance and overall increases the reliability and specificity of the analysis. However, the process of modification with biorecognition elements (so-called functionalization) may influence the resulting antifouling properties. The extent of these effects concerning both functionalization procedures potentially changing the surface architecture and properties, and the physicochemical properties of anchored biorecognition elements, remains unclear and has not been summarized in the literature yet. This critical review summarizes these key functionalization aspects with respect to diverse antifouling architectures showing low or ultra-low fouling quantitative characteristics in complex biological media such as bodily fluids or raw food samples. The subsequent discussion focuses on the impact of functionalization on fouling resistance. Furthermore, this review discusses some of the drawbacks of available surface sensitive characterization methods and highlights the importance of suitable assessment of the resistance to fouling.
Functional polymer coatings that combine the ability to resist non-specific fouling from complex media with high biorecogni-tion element (BRE) immobilization capacity represent an emerging class of new functional materials for a number of bioanalyti-cal and biosensor technologies for medical diagnostics, security, and food safety. Here we report on a random copolymer brush surface - poly(CBMAA-ran-HPMAA) - providing high BRE immobilization capacity while simultaneously exhibiting ul-tralow-fouling behavior in complex food media. We demonstrate that both the functionalization and fouling resistance capabili-ties of such copolymer brushes can be tuned by changing the surface contents of the two monomer units: non-ionic N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxy-functional zwitterionic carboxybetaine methacrylamide (CBMAA). It is demonstrated that the resistance to fouling decreases with the surface content of CBMAA; poly(CBMAA-ran-HPMAA) brushes with CBMAA molar content up to 15 mol% maintain excellent resistance to fouling from a variety of homogenized foods (hamburger, cucumber, milk, and lettuce) even after covalent attachment of BREs to carboxy-groups of CBMAA. The poly(CBMAA 15 mol%-ran-HPMAA) brushes functionalized with antibodies are demonstrated to exhibit fouling resistance from food samples by up to three orders of magnitude better when compared with the widely used low-fouling carboxy-functional oligo(ethylene glycol) (OEG)-based alkanethiolate self-assembled monolayers (AT SAMs) and furthermore, by up to two orders of magnitude better when compared with the most successful ultralow-fouling biorecognition coatings - poly(carboxybetaine acrylamide), poly(CBAA). Using model SPR detections of foodborne bacterial pathogens in homogenized foods it is also demonstrated that the antibody-functionalized poly(CBMAA 15 mol%-ran-HPMAA) brush exhibits superior biorecognition properties over the poly(CBAA).
We reported here two novel electrochemical impedance spectroscopy biosensors were developed for the first time for highly sensitive quantification of matrix metalloproteinase-14 (MMP-14) based on binding interaction between hemopexin-like domain (PEX) of MMP-14 (PEX-14) and its inhibitory peptides. Specific inhibitory peptides (IVSC or ISC) inhibiting homodimerization or heterodimerization of MMP-14 was first self assembled on the surface of gold electrode and blocked with 6-mercapto-1-hexanol on a gold electrode surface used as IVSC or ISC modified biosensor, respectively. IVSC modified biosensor can be used for detection of MMP-14 by using the direct IVSC-MMP-14 interaction inhibiting MMP-14 homodimerization as well as ISC modified biosensor for indirect detection of MMP-14 via PEX-14 mediated peptide-MMP-14 binding. The electron transfer resistance (Ret) of biosensor was monitored to measure MMP-14 using Fe(CN)63-/4- as probe. The increase of the Ret of the biosensors are linear with the concentration of MMP-14 in the range from 1 μg L-1 to 10 μg L-1 with detection limit of 0.19 μg L-1 for IVSC modified biosensor and 0.1 ng L-1 to 50 ng L-1 with detection limit of 7 ng L-1 for ISC modified biosensor. This work demonstrates that probing the interaction between peptide inhibitor and PEX of MMPs represents a novel approach to assess MMPs-mediated cancer dissemination.
Herein, an electrochemical immunosensor capable of detecting targets in human serum with ultralow fouling and high sensitivity was successfully constructed through a click reaction. One designed biotinylated peptide was associated with the other peptide functionalized with biotin and azide groups at its two terminals, to form a branched antifouling peptide through the streptavidin-biotin affinity interaction, and it was attached to the electrode to construct an antifouling interface. Then, the exposed azide group on the branched peptide was used to link the antibody modified with 5’-dibenzocyclooctyne (DBCO), and the immunosensor was thus constructed with the aid of the click reaction between the azide group and the DBCO. The developed antifouling immunosensor demonstrated a wide linear response range for carcinoembryonic antigen (CEA) and a low limit of detection of 40 fg mL⁻¹, which is much superior to the sensing performance of the immunosensor prepared through the normal EDC/NHS method. The immunosensor was capable of assaying CEA in undiluted serum samples with satisfying accuracy when compared with a clinically approved method. This work offered an effective strategy to develop various biosensors through the conjugation of antifouling peptides with different antibodies.
Zwitterionic peptides are facile low-fouling compounds for environmental applications as they are biocompatible and fully biodegradable as their degradation products are just amino acids. Here, a set of histidine (H) and glutamic acid (E), as well as lysine (K) and glutamic acid (E) based peptide sequences with zwitterionic properties were synthesized. Both oligopeptides (KE)4K and (HE)4H were synthesized in d and l configurations to test their ability to resist the nonspecific adsorption of the proteins lysozyme and fibrinogen. The coatings were additionally tested against the attachment of the marine organisms Navicula perminuta and Cobetia marina as well as the freshwater bacterium Pseudomonas fluorescens on the developed coatings. While the peptides containing lysine performed better in protein resistance assays and against freshwater bacteria, the sequences containing histidine were generally more resistant against marine organisms. The contribution of amino acid-intrinsic properties such as side chain pKa values and hydrophobicity, as well as external parameters such as pH and salinity of fresh water and seawater on the resistance of the coatings is discussed. In this way, a detailed picture emerges as to which zwitterionic sequences show advantages in future generations of biocompatible, sustainable, and nontoxic fouling release coatings.
Sensors have been applied in a broad range of fields, but they are limited to surface fouling caused by real complex samples, which severely influence the target recognition leading to poor reliability and stability. Therefore, it is necessary to develop antifouling sensing platforms for maintaining sensors performance. For this purpose, this section provides a comprehensive review to discuss the mechanisms of antifouling surfaces, the influence factors, as well as the modification strategies. Furthermore, kinds of antifouling materials are systematically introduced with their properties and applications in sensors. And the challenges and perspectives of antifouling materials for sensors are also discussed here.
Due to the limited clinical utility of individual biomarkers, there is growing recognition of the need for combining multiple biomarkers as a panel to improve the accuracy and efficacy of disease diagnosis and prognosis. The conventional method to detect multiple analyte species is to construct a sensor array, which consists of an array of individual selective probes for different species. In this work, by using cancer biomarker matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) as model analytes and functionalized nanographene oxide (nGO) as a sensing element, we developed a multiplexing fluorescence sensor in a nonarray format for simultaneous measurement of the activities of multiple proteases. The constructed nGO-based biosensor was rapid, sensitive, and selective and was also utilized for the successful profiling of ADAMs/MMPs in simulated serum samples. Furthermore, we showed that joint entropy and programming could be utilized to guide experiment design, especially in terms of the selection of a subset of proteases from the entire MMPs/ADAMs family as an appropriate biomarker panel. Our developed nGO-based multiplex sensing platform should find useful application in early cancer detection and diagnosis.
An electrochemical peptide-based biosensor is described for the determination of matrix metalloproteinase-14 (MMP-14). The method is base on MMP-14-induced cleavage of a specific peptide carrying a ferrocene group (CK11-Fc) placed on a gold electrode. CK11-Fc was first self-assembled on the electrode and the residual surface was blocked with 6-mercapto-1-hexanol. When MMP-14 was present, it specifically cleaves the CK11-Fc. Hence, Fc leaves the electrode and this causes a decrease of the electrochemical signal, best measured at 500 mV (vs. Ag/AgCl) in Tris buffer of pH 7.4. The signal decreases linearly in the 0.2 ng L⁻¹ to 0.9 ng L⁻¹ MMP-14 concentration range, and the detection limit is 0.1 ng L⁻¹ (RSD = 5.7 %). The biosensor was successfully applied to the determination of MMP-14 in overexpressed tumor cells (type MCF-7). Conceivably, the work has a wide potential by exploiting such peptide cleavage approaches to construct biosensors for MMPs.
Zwitterionic peptides are great candidates as antifouling coating materials in many biomedical applications. We investigated the structure and antifouling properties of surface tethered zwitterionic peptide monolayers with different peptide chain lengths and charge distributions using a combination of surface plasma resonance (SPR), atomic force microscopy (AFM), and all atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that zwitterionic peptides with more zwitterionic lysine (K) and glutamic acid (E) repeating units exhibit better antifouling performance. The block charge distributions of the positive and negative charges in the peptides (having multiple positive charges next to the same amount of negative charges) although affecting the structure of the peptide molecules, do not significantly change the antifouling properties of the peptide monolayers in the solutions containing monovalent ions. However, divalent cations, Ca2+ and Mg2+, in solution can significantly alter the structure and lower the antifouling performance of the zwitterionic peptide monolayers, especially with the sequences of block charges. All atomistic MD simulations quantitatively reveal that the divalent cations in solution lead to more inter-chain electrostatic crosslinks between peptide chains, especially for peptides with block charges, which causes dehydration of the zwitterionic peptides and diminishes their antifouling performances.
m-Aminobenzoic acid (ABA) is one popular derivative of highly conductive monomer of aniline, which contains a carboxyl (COOH) group in its skeleton that is beneficial to various bio-interface and bio-analysis. Hence, Poly(m -aminobenzoic acid) (PABA) membrane was firstly electrochemical deposited onto bare electrode surface using a straightforward cyclic voltammetry (CV) method. PABA membrane exhibited excellent electrochemical stability and apparent wrinkle morphology that could effectively enhance response signal and immensely increase specific surface area of electrode. Next, PABA membrane was decorated with well-designed hairpin aptamer and preferred antifouling peptide in sequence to construct a two-layer architectural bio-interface that could present both sensitive target recognition capability and excellent antifouling ability. Benefiting from the electrodeposited PABA membrane to enhance electrochemical response signal, the developed biosensor performed excellent sensitivity toward target protein, meanwhile, associated with good selectivity and reproducibility attributing to the favored peptide that was able to decline nonspecific protein adsorption and improve target recognition.
A luminescent biosensor has been developed for matrix metalloproteinase 9 (MMP-9) assay based on a selective interaction between Ir(III) solvent complex with histidine-rich peptide, which avoids the complicated double labeling...
Zwitterionic peptides were anchored to a conducting polymer of citrate doped poly(3,4-ethylenedioxythiophene) (PEDOT) via the nickel cation coordination, and the obtained peptide modified PEDOT, with excellent antifouling ability and good conductivity, was further used for the immobilization of a DNA probe to construct an electrochemical biosensor for the breast cancer marker BRCA1. The DNA biosensor was highly sensitive (with detection limit of 0.03 fM) and selective, and it was able to detect BRCA1 in 5% (V/V) human plasma with satisfying accuracy and low fouling. The marriage of antifouling and biocompatible peptides with conducting polymers opened a new avenue to construct electrochemical biosensors capable of assaying targets in complex biological media with high sensitivity and without biofouling.
An electro-active nanoscale composite was engineered to serve as an alternative to monolayers in a capacitive biosensing platform. An electrochemical impedance-derived spectroscopic method was applied to analyse the response of this nanoscale (c.a. 9 nm thickness) composite to detect C-reactive protein. The composite was successfully assembled over a conductive electrode and built up by conjugating graphene oxide sheets into a self-assembled 11-ferrocenyl-undecanethiol structure, forming a molecular composite concomitantly coated with zwitterionic methacrylate-based monomers that served as the non-fouling component of the composite. Finally, the functionalization of a composite with receptors allowed the successful testing of its electro-analytical ability to detect C-reactive protein in both phosphate-buffered saline and neat human serum samples.
Self-assembled monolayers (SAMs) are widely used in science and engineering, and recent progress has demonstrated the utility of zwitterionic peptides with alternating lysine (K) and glutamic acid (E) residues for antifouling purposes. Aiming at developing a peptide-based fouling-resistant SAM suitable for presentation of surface-attached pheromones for barnacle larvae, we have investigated five different peptide SAMs, where four are based on the EK motif, and the fifth was designed based on general principles for fouling resistance. The SAMs were formed by self-assembly onto gold substrates via cysteine residues on the peptides, and formation of SAMs was verified via ellipsometry, wettability, infrared reflection-absorption spectroscopy and cyclic voltammetry. Settlement of cypris larvae of the barnacle Balanus (=Amphibalanus) amphitrite, the target of pheromone studies, was tested. SAMs were also subjected to fouling assays using protein solutions, blood serum, and the bacterium Mycobacterium marinum. The results confirm the favourable antifouling properties of EK-containing peptides in most of the assays, although this did not apply to the barnacle larvae settlement test, where settlement was low on only one of the peptide SAMs. The one peptide that had antifouling properties for barnacles did not contain a pheromone motif, and would not be susceptible to degredation by common serine proteases. We conclude that the otherwise broadly effective antifouling properties of EK-containing peptide SAMs is not directly applicable to barnacles, and that great care must be exercised in the design of peptide-based SAMs for presentation of barnacle-specific ligands.
The ability to monitor biomolecular reactions in a real-time and label free manner is nearly synonymous with the surface plasmon resonance (SPR) technique. At its basis, SPR relies on the interaction of an incoming light source with a thin metallic film in close contact with a prism or grating, which, under the correct experimental conditions, allows for the propagative oscillation of the conduction electrons at the surface of the metallic material. The oscillating, electromagnetic fields are extremely sensitive to small changes in the refractive index within the dielectric near the sensing interface, where various binding events and interactions can be detected. Historically, SPR has seen its predominant use within pharmaceutical research, where commercial instrumentation has been employed for drug discovery, antibody characterization, and studies of protein/enzyme inhibition. There have recently been a number of excellent reviews focused on the various applications in which SPR spectroscopy and its derivatives can be employed, and therefore, within this review we discuss SPR from a design perspective, covering the various instrumental and interfacial aspects that have been created and optimized only within the past couple of years in order to highlight the increased accessibility of this platform to a wider range of scientific researchers and disciplines.
Advances in the fabrication of bioelectrochemical interfaces has provide us with something close to molecular level control over both the immobilisation of biomolecules on an electrode and the environment around which they are immobilised. This control can be both lateral, by changing the ratio of components in a mixed monolayer, and vertical using rigid self-assembling molecules. The most important advances in the last few years are the development of methods that allow amperometric systems to operate in complex biological fluids and new methods of characterising the bioelectrochemical interfaces at the molecular level at which they are designed. The purpose of this Current Opinion review is to outline the advances in low impedance antifouling coatings on electrodes for bioelectrochemistry and the use of cutting edge fluorescence microscopy to characterise the function of these interfaces in the environments they are intended to be used in with high spatial resolution.
Gold-coated nanodisk arrays of nearly micron periodicity are reported that have high figure of merit (FOM) and sensitivity necessary for plasmonic refractometric sensing, with the added benefit of suitability for surface-enhanced Raman scattering (SERS), large-scale microfabrication using standard photolithographic techniques and a simple instrumental setup. Gold nanodisk arrays are covered with a gold layer to excite the Bragg modes (BM), which are the propagative surface plasmons localized by the diffraction from the disk array. This generates surface-guided modes, localized as standing waves, leading to highly confined fields confirmed by a mapping of the SERS intensity and numerical simulations with 3D finite element method. The optimal gold-coated nanodisk arrays are applied for refractometric sensing in transmission spectroscopy with better performance than nanohole arrays and they are integrated to a 96-well plate reader for detection of IgY proteins in the nanometer range in PBS. The potential for sensing in biofluids is assessed with IgG detection in 1:1 diluted urine. The structure exhibits a high FOM of up to 46, exceeding the FOM of structures supporting surface plasmon polaritons and comparable to more complex nanostructures, demonstrating that subwavelength features are not necessary for high-performance plasmonic sensing.
Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-peptide microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, 3, 7, 9, 14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs, and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL-1 for MMP-2, 60 pg mL-1 for MMP-3, 0.22 pg mL-1 for MMP-7, 102 fg mL-1 for MMP-9 and 0.68 ng mL-1 for MMP-14, respectively. The practicability of the FRET-peptide microarray-based MEF assay is demonstrated by profiling multiplexed MMPs activities of various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) patients and thyroid nodules (TN) patients, and satisfactory results are obtained.
Optical biosensors such as those based on surface plasmon resonance (SPR) are a key analytical tool for understanding biomolecular interactions and function as well as the quantitative analysis of analytes in a wide variety of settings. The advent of portable SPR instruments enables analyses in the field. A critical step in method development is the passivation and functionalisation of the sensor surface. We describe the assembly of a surface of thiolated oleyl ethylene glycol/biotin oleyl ethylene glycol and its functionalisation with streptavidin and reducing end biotinylated heparin for a portable SPR instrument. Such surfaces can be batch prepared and stored. Two examples of the analysis of heparin-binding proteins are presented. The binding of fibroblast growth factor 2 and competition for the binding of a heparan sulfate sulfotransferase by a library of selectively modified heparins and suramin, which identify the selectivity of the enzyme for sulfated structures in the polysaccharide and demonstrate suramin as a competitor for the enzyme’s sugar acceptor site. Heparin functionalised surfaces should have a wide applicability, since this polysaccharide is a close structural analogue of the host cell surface polysaccharide, heparan sulfate, a receptor for many endogenous proteins and viruses.
Monitoring the response of patients undergoing chemotherapeutic treatments is of great importance to predict remission success, avoid adverse effects and thus, maximise the patients’ quality of life. In the case of leukemia patients treated with E. coli L-asparaginase, monitoring the immune response by the detection of specific antibodies to L-asparaginase in the serum of patients can prevent extended immune response to the drug. Here, we developed a surface plasmon resonance (SPR) biosensor to rapidly detect anti-asparaginase antibodies directly in patients’ sera, without requiring sample pre-treatment or dilution. A direct assay with SPR sensing to detect anti-asparaginase antibodies exhibited a limit of detection of 500 pM and a high sensitivity range between 100 nM and 1 μM in pooled and undiluted human serum from a commercial source. While the SPR assay showed excellent reproducibility (12% CV) in pooled serum, challenges were encountered upon analyzing clinical samples due to high sample-to-sample variability in color and turbidity and, in all likelihood, in composition. As a result, direct detection in clinical samples was unreliable due to factors that may generally affect assays based on plasmonic detection. Addition of a secondary detection step overcame sample variability due to bulk differences in patients’ sera. By those means, the SPR biosensor was successfully applied to the analysis of clinical samples from leukemia patients undergoing asparaginase treatments and the results agreed well with the standard ELISA assay. Monitoring antibodies against drugs is common such that this type of sensing scheme could serve to monitor a plethora of immune responses in sera of patients undergoing treatment.
The design and application of sensors for monitoring biomolecules in clinical samples is a common goal of the sensing research community. Surface plasmon resonance (SPR) and other plasmonic techniques such as localized surface plasmon resonance (LSPR) and imaging SPR are reaching a maturity level sufficient for their application in monitoring biomolecules in clinical samples. In recent years, the first examples for monitoring antibodies, proteins, enzymes, drugs, small molecules, peptides, and nucleic acids in biofluids collected from patients afflicted with a series of medical conditions (Alzheimer’s, hepatitis, diabetes, leukemia, and cancers such as prostate and breast cancers, among others) demonstrate the progress of SPR sensing in clinical chemistry. This Perspective reviews the current status of the field, showcasing a series of early successes in the application of SPR for clinical analysis and detailing a series of considerations regarding sensing schemes, exposing issues with analysis in bioflui...
Peptidomimetic polymers consisting of poly-N-substituted glycine oligomers (polypeptoids) conjugated to biomimetic adhesive polypeptides were investigated as antifouling surface coatings. The polymers were immobilized onto TiO(2) surfaces via an anchoring peptide consisting of alternating residues of 3,4-dihydroxyphenylalanine (DOPA) and lysine. Three polypeptoid side-chain compositions were investigated for antifouling performance and stability toward enzymatic degradation. Ellipsometry and XPS analysis confirmed that purified polymers adsorbed strongly to TiO(2) surfaces, and the immobilized polymers were resistant to enzymatic degradation as demonstrated by mass spectrometry. All polypeptoid-modified surfaces exhibited significant reductions in adsorption of lysozyme, fibrinogen and serum proteins, and were resistant to 3T3 fibroblast cell attachment for up to seven days. Long-term in vitro cell attachment studies conducted for six weeks revealed the importance of polypeptoid side-chain composition, with a methoxyethyl side chain providing superior long-term fouling resistance compared to hydroxyethyl and hydroxypropyl side chains. Finally, attachment of both gram-positive and gram-negative bacteria for up to four days under continuous-flow conditions was significantly reduced on the polypeptoid-modified surfaces compared to unmodified TiO(2) surfaces. The results reveal the influence of polypeptoid side-chain chemistry on short-term and long-term protein, cell and bacterial fouling resistance.
Using a heterotelechelic poly(ethylene glycol) (PEG) possessing a mercapto group at one end and an acetal group at the other end (acetal-PEG-SH), the authors constructed a reactive PEG tethered-chain surface on a surface plasmon sensor (SPR) gold chip for biosensing with high sensitivity. Nonspecific bovine serum albumin adsorption on the PEG tethered-chain surface was significantly influenced by the density of the PEG chain, and was almost completely suppressed by increasing the PEG density through the repetitive treatment of the chip surface with acetal-PEG-SH. The PEG density was increased even more by adding an underbrushed layer made of shorter-PEG-SH-chain molecules (2 kDa, hereafter 2k) to the surface made of longer-PEG-SH-chain molecules (5 kDa, hereafter 5k). SPR measurement then gave an estimate of the adsorption of a series of proteins with varying sizes and isoelectric points on the PEG chain surface having an underbrushed layer (PEG5k/2k surface). As compared to other SPR surfaces, viz., a commercial carboxymethyl dextran and conventional PEG5k tethered-chain surface without an underbrushed layer, the mixed PEG5k/2k surface showed almost complete inhibition of the nonspecific adsorption not only of high-molecular-weight proteins but also of low-molecular-weight peptides.
Leucocyte (L)-selectin can be proteolytically cleaved in the membrane proximal extracellular region to yield a soluble fragment that contains the functional lectin and epidermal growth factor domains. A variety of stimuli are known to stimulate L-selectin shedding including chemoattractants, phorbol esters, and L-selectin cross-linking; however, the enzymes that regulate L-selectin expression are not characterized. In this study we have used phorbol ester to stimulate endoproteolytic release of L-selectin and identified a major role for a cell surface metalloproteinase (L-selectin sheddase) in this process.
The hydroxamic acid-based inhibitor of zinc-dependent matrix metalloproteinases Ro 31-9790 completely prevented shedding of cell surface L-selectin from leucocytes in mouse, rat, and man. L-selectin was susceptible to cleavage by known matrix metalloproteinases. Recombinant human fibroblast collagenase (MMP1) reduced the number of L-selectin-positive lymphocytes to a similar extent as phorbol ester activation, and stromelysin (MMP3) had a partial effect on L-selectin expression. Gelatinases A (MMP2) and B (MMP9) were without effect. Lymphocytes did not express fibroblast collagenase or stromelysin at the cell surface, and tissue inhibitor of metalloproteinases (TIMP) did not affect L-selectin levels. L-selectin sheddase was not detected in media harvested from phorbol ester-stimulated lymphocytes and was only able to cleave L-selectin in the cis but not the trans configuration.
These results suggest that endoproteolytic release of L-selectin from the leucocyte surface is mediated by a metalloproteinase (L-selectin sheddase), which is distinguishable from known matrix metalloproteinases. Understanding the regulation of L-selectin sheddase will be critical for controlling leucocyte migration from the blood.
Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably upregulated in epithelial cancers and are key agonists in angiogenesis, invasion and metastasis. Yet most MMPs are secreted not by the cancer cells themselves, but by stromal cells within and around the tumor mass. Because the stromal environment can influence tumor formation, and because MMPs can alter this environment, MMPs may also contribute to the initial stages of cancer development. Several recent studies in MMP-overexpressing and MMP-deficient mice support this possibility, but have required carcinogens or pre-existing oncogenic mutations to initiate tumorigenesis. Here we review the spontaneous development of premalignant and malignant lesions in the mammary glands of transgenic mice that express an autoactivating form of MMP-3/stromelysin-1 under the control of the whey acidic protein gene promoter. These changes were absent in nontransgenic littermates and were quenched by co-expression of a human tissue inhibitor of metalloproteinases-1 (TIMP-1) transgene. Thus by altering the cellular microenvironment, stromelysin-1 can act as a natural tumor promoter and enhance cancer susceptibility.
The novel modification of gold surfaces with a biotinylated polypeptide multilayer formed by nonspecific electrostatic adsorption was designed for the SPR sensing platform. The multilayer of polypeptides (poly-L-lysine and poly(aspartic acid)) was prepared onto a negatively charged self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) on gold film. The polypeptide multilayer was functionalized with biotin by directly grafting to poly(succinimide) (PSI). The specific binding of avidin to the biotinylated polypeptide multilayer (DS 2.7%) resulted in a shift in the refractive index of 0.003±9×10-4 with a surface density of 2.9 ng/mm2. This multilayer surface was found to provide a simple and effective method for the SPR sensing.
Gold surfaces intended for surface plasmon resonance sensors have been derivatized with a flexible matrix composed of carboxymethylated dextran to provide fast and efficient coupling of proteins and other ligands at low concentrations using the principles of ionic pre-concentration; these hydrophilic and protein compatible hydrogel surfaces allow increased immobilization capacity as compared to monolayer based coatings, for applications in biospecific interaction analysis.
A surface plasmon resonance-based immunosensor was developed by modifying the surface of a thin polyion complex film consisting of poly-l-lysine and poly-styrenesulfonate. The suppression of non-specific protein adsorption was investigated by changing the blend ratio of the two polymers. The protein adsorption fell to less than 10% that of an unmodified surface when the poly-l-lysine to ratio was 4:6. The binding constant between immobilized B-type natriuretic peptide on the polyion complex film and its antibody was calculated to be 1.1×108 (M−1), which is a satisfactory level for immuno-affinity on a solid surface. We also obtained stable and reproducible responses without losing the affinity on the polyion complex film.
Fmoc-histidine derivatives protected with 2,6-dimethoxybenzoyl (2,6-Dmbz) units, either on the τ- (1) or on the π-position (2) of the imidazole residue, have been prepared and applied to the synthesis of the dipeptide Fmoc-His(2,6-Dmbz)-Val-OMe, with high coupling efficiencies (99 and 95%, respectively) and low racemization (0.3 and 0.1%, respectively), as ascertained by HPLC analysis on the fully protected dipeptides. In addition, the hexapeptide H-Ala-Ser-Val-His-Val-Phe-OH (I) has been synthesized on a solid support employing the novel building blocks 1 and 2 as well as commercially available Fmoc-His(τ-Trt)-OH (3). The crude deprotected products were analyzed by reverse-phase HPLC and mass spectrometry, which indicated that when the τ-protected derivatives 1 and 3 were employed the products were of comparable quality (93−94% major peak). The hexapeptide synthesized with the π-protected histidine 2 was contaminated with a product corresponding to 2,6-Dmbz-His-Val-Phe-OH (12), which was probably formed through the intramolecular acyl transfer of a 2,6-Dmbz unit from the π-nitrogen atom to the α-amino group of the N-terminal histidine unit upon removal of the Fmoc group. This side reaction was insignificant (less than 0.5%) when using the τ-protected derivative 1. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
This paper describes a procedure for preparing mixed self-assembled monolayers (mixed SAMs) on gold that resist the nonspecific adsorption of proteins from solution. This method was tested using α-amino derivatives of ω-hydroxy- and ω-methoxy-oligo(ethylene glycols): H2N(CH2CH2O)nCH3 and H2N(CH2CH2O)nH (n = 3, 6). Mixed SAMs were prepared by allowing these amines to react with a SAM presenting interchain carboxylic anhydride groups. The resistance of the resulting surfaces to adsorption of several proteinscarbonic anhydrase (EC 4.2.1.1), ribonuclease A (EC 3.1.27.5), lysozyme (EC 3.2.1.17), and fibrinogenwas examined using surface plasmon resonance (SPR) spectroscopy. These mixed SAMs resist the nonspecific adsorption of proteins approximately as effectively as single-component SAMs prepared using the conventional method involving chemisorption of oligo(ethylene glycol)-terminated alkanethiols on gold. Characterization of the mixed SAM that presents a 1:1 mixture of −OCNH(CH2CH2O)6CH3 and CO2H/CO2- groups by polarized infrared external reflectance spectroscopy indicates that the ethylene glycol units are in an amorphous conformation. A model surface for use in studies of biospecific adsorption was synthesized by reacting the anhydride groups with a mixture of H2N(CH2CH2O)6H and H2N(CH2CH2O)6CH2CONH(CH2)6NHCOC6H4SO2NH2; the resulting system was examined for its ability to bind bovine carbonic anhydrase by SPR. The values of the relevant constants were koff = 0.0054 s-1, kon = 13 000 M-1 s-1, and Kd = 0.42 μM. These values agree with values obtained by other means. The reaction of amines with SAMs that present interchain carboxylic anhydrides provides an experimentally simple route to the formation of mixed SAMs that resist the nonspecific adsorption of proteins or that adsorb a protein of interest biospecifically.
A simple but quantitative mathematical formalism for interpretation of surface plasmon resonance (SPR) signals from adsorbed films of a wide variety of structures is presented. It can be used to estimate adsorbed film thicknesses, surface coverages, or surface concentrations from the SPR response over the entire range of film thicknesses without relying on calibration curves of response versus known thicknesses or surface concentrations. This formalism is compared to more complex optical simulations. It is further tested by (1) calibrating the response of two SPR spectrometers to changes in bulk index of refraction, (2) using these calibrations with this formalism to predict responses to several well-characterized adlayer structures (alkanethiolates and serum albumin on gold, propylamine on COOH-functionalized gold), and then (3) comparing these predictions to measured SPR responses. Methods for estimating the refractive index of the adlayer material are also discussed. Detection limits in both bulk and adsorption-based analyses are discussed. The planar system used here has a detection limit of 0.003 nm in average film thickness for adsorbates whose refractive index differs from that of the solvent by only 0.1. The temperature sensitivities of these two SPR spectrometers are characterized and discussed in terms of detection limits.
This paper describes the use of surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) to determine the characteristics of functional groups that give surfaces the ability to resist the nonspecific adsorption of proteins from solution. Mixed SAMs presenting different functional groups were prepared for screening using a synthetic protocol based on the reaction of organic amines with a SAM terminated by interchain carboxylic anhydride groups. Surfaces that presented derivatives of oligo(sarcosine), N-acetylpiperazine, and permethylated sorbitol groups were particularly effective in resisting the adsorption of proteins. Incorporation of these groups into single-component SAMs resulted in surfaces that are comparable to (but slightly less good than) single-component SAMs that present oligo(ethylene glycol) in their ability to resist the adsorption of proteins. In the group of surfaces examined, those that resisted the adsorption of proteins had the following properties: they were hydrophilic; they contained groups that were hydrogen-bond acceptors but not hydrogen-bond donors; and they were overall electrically neutral.
This paper demonstrates that surface plasmon resonance (SPR) spectroscopy can be used to measure the nonspecific adsorption of proteins to self-assembled monolayers (SAMs) of alkanethiolates on gold in situ and in realtime. Mixed SAMs comprising hexa(ethylene glycol) and methyl groups that have values of (chi Me) < 0.5 resisted the adsorption of four test proteins: RNase A, lysozyme, fibrinogen, and pyruvate kinase. These four proteins adsorbed irreversibly to surfaces having values of (chi Me) > 0.5: the amount of adsorbed protein correlated with (chi Me). The initial rate for adsorption of fibrinogen to a methyl-terminated SAM ((chi Me) = 1.0) followed first-order kinetics. The combination of SAMs and SPR described here is particularly well suited for investigations of the interactions of proteins with structurally well-defined organic surfaces.
The secondary structure of the synthetic HELELELELELC peptide sequence was studied by molecular dynamics simulations at two different pH values. At pH 2, the α helix conformation is stable over 200 ns, but at pH 7, a random coil is irreversibly obtained after 70 ns, due to the electrostatic repulsions between the negatively charged carboxylate groups of the glutamic acid residues. The sensitivity of the structure to the pH was used to fabricate pH-responsive monolayers of peptide by self-assembly. Electrochemical measurements were performed to assess the properties of the monolayer as a function of the pH, using redox couples either dissolved in solution or attached to the peptide by coordination at the amino-terminal histidine side chain. In this latter case, no discernible electron transfer to/from the redox center took place at pH 6.9 because of the extended conformation adopted by the peptide. By contrast, the rate of electron transfer became increasingly faster upon decrease of the pH, with rate constants of 78, 110, and 230 s−1 determined at pH 3.0, 2.0, and 1.0.
This paper describes the use of surface plasmon resonance spectroscopy and self-assembled monolayers (SAMs) of alkanethiols on gold to evaluate the ability of surfaces terminating in different combinations of charged groups to resist the nonspecific adsorption of proteins from aqueous buffer. Mixed SAMs formed from a 1:1 combination of a thiol terminated in a trimethylammonium group and a thiol terminated in a sulfonate group adsorbed less than 1% of a monolayer of two proteins with different characteristics: fibrinogen and lysozyme. Single-component SAMs formed from thiols terminating in groups combining a positively charged moiety and a negatively charged moiety were also capable of resisting the adsorption of proteins. Single-component SAMs presenting single charges adsorbed nearly a full monolayer of protein. The amount of protein that adsorbed to mixed zwitterionic SAMs did not depend on the ionic strength or the pH of the buffer in which the protein was dissolved. The amount of protein that adsorbed to single-component zwitterionic SAMs increased as the ionic strength of the buffer decreased; it also decreased as the pH of the buffer increased (at constant ionic strength). Single-component zwitterionic SAMs composed of thiols terminating in N,N-dimethyl-amino-propane-1-sulfonic acid (-N + (CH3)2CH2CH2CH2SO3 -) groups were substantially more effective at resisting adsorption of fibrinogen and lysozyme from buffer at physiological ionic strength and pH than single-component zwitterionic SAMs composed of thiols terminating in phosphoric acid 2-trimethylamino-ethyl ester (-OP(O)2 -OCH2CH2N + (CH3)3). Several of these zwitterionic SAMs were comparable to the best known systems for resisting nonspecific adsorption of protein.
The modification of alkanethiol monolayers via amide and ester formation reactions is demonstrated as a methodology for attaching monolayer and submonolayer coverages of specific chemical species onto metal electrodes. A monolayer of 11-mercaptoundecanoic acid (HSC10H20COOH) is used as a surface bifunctional linking agent on polycrystalline gold surfaces. The reaction of these adsorbed molecules with gaseous thionyl chloride converts the carboxylic acid group to an acid chloride which can then be reacted further with an amine or alcohol to form an amide or ester linkage. The attachment of alkyl, aromatic, and electrochemically active species to the surface is reported to demonstrate the versatility of this surface derivatization chemistry. Polarization modulation Fourier transform infrared (PM-FTIR) spectroscopy is employed as the primary method for monitoring the chemical structure of the adsorbed monolayers, and additional X-ray photoelectron spectroscopy (XPS) and electrochemical measurements are used to verify the elemental composition, oxidation state, and surface coverage of the adsorbed molecules.
BACKGROUND
The relationship between serum levels of matrix metalloproteinase-2 (MMP-2) and MMP-3 and recurrence in patients with urothelial carcinoma after complete resection was studied to determine whether the enzymes could be a new predictor of recurrence.METHODS
Serum levels of MMP-2 and MMP-3 in 146 healthy controls, 52 patients with superficial (noninvasive) and 35 patients with advanced (invasive or metastatic) bladder carcinoma, and 30 patients with advanced upper urothelial carcinoma (renal pelvis and ureter) were measured by a one-step sandwich enzyme assay.RESULTSAmong the 53 patients with advanced urothelial carcinoma who underwent complete resection, the 1- and 3-year disease free survival rates of patients with elevated serum levels of either or both of the enzymes were 51% and 19%, respectively, whereas those of patients with normal serum levels of these enzymes were much higher, 86% and 79%, respectively (P < 0.005). In pT2N0M0 and pT3N0M0 patients who underwent complete resection, the recurrence rate in those with preoperative elevated serum levels of either or both of the enzymes was significantly higher than that in patients with normal levels (75% vs. 8%; P < 0.001). The 1- and 3-year disease free survival rates of pT2N0M0 and pT3N0M0 patients with elevated serum levels of either or both of the enzymes were 55% and 21%, respectively, which was significantly lower than that of patients with normal levels (92%; P < 0.001).CONCLUSIONS
The results of this study indicate that the elevation of serum levels of either MMP-2 or MMP-3 or both could be new predictors of recurrence in patients with advanced urothelial carcinoma after complete resection. Cancer 1996;78:2379-87.
Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are involved in important processes of tumor invasion and metastasis. in the study presented, matrix metalloproteinase 1 (MMP1) and 3 (MMP3), the tissue inhibitor of metalloproteinase 1 (TIMP1) and the complex MMP1/TIMP1 were measured by ELISA tests specific for these proteins in blood plasma. These components have been investigated in prostate cancer patients (PCa) with metastases (n = 18; T2, 3, 4 pN1, 2M1), prostate cancer patients without metastases (n = 29; T2, 3 pN0M0), patients with benign prostate hyperplasia (BPH; n = 29) and in healthy men (n = 35). Mean values of MMP1 and of the complex MMP1/TIMP1 were not different among the four groups. The mean values of MMP3 and especially TIMP1 were significantly higher in prostate cancer patients with metastases compared with controls, BPH patients and prostate cancer patients without metastases. Ten of these 18 patients had TIMP1 levels higher than the upper reference limit. TIMP1 concentrations correlate to the tumor stage but not to the tumor grade. These results indicate, that TIMP1 could be an potential marker for metastases in prostate cancer patients.
Control of nonspecific interactions between bioanalytical surfaces and proteins in the analyte is critical in the design of biosensor systems. Here we explore poly(propylene sulfide-block-ethylene glycol) (PPS-PEG) di- and triblock copolymer adlayers on gold to gain such control. Chemisorption of the PPS block permits a simple dip-and-rinse coating process. We synthesized different architectures of di- and triblock copolymers, varying the molecular weight of PEG between 1.1 and 5 kDa while keeping the PPS block constant at around 4 kDa, thus permitting systematic variations in ethylene glycol surface density in the adlayer. A simple dip-and-rinse process was used to produce PPS-PEG adlayers on gold substrates, which were characterized with surface plasmon resonance (SPR) and further confirmed by existing variable angle spectral ellipsometry (VASE), and X-ray photoelectron spectroscopy (XPS). Crowding in the PPS chemisorbed layer seemed to limit the polymer adsorption process. Subsequent exposure of PPS-PEG adlayers to protein adsorption (human serum albumin at 1 mg/mL or full-concentration human serum) was monitored with in situ SPR. Protein adsorption can be reduced up to 97% for human serum albumin and up to 96% for blood serum relative to bare gold substrates. Triblock copolymers were more effective than corresponding diblocks. The possibility to render gold surfaces bioinert is the basis for application in bioanalytical devices.
Recently, the modification of gold surfaces with covalently bound carboxylated dextran was described, for use in biospecific interaction studies in an analytical system based on surface plasmon resonance (SPR). In this study, the antigen binding immunoreactivity of antibodies immobilized on the modified surfaces was investigated with the SPR-based system. Four different monoclonal antibodies specific against the antigens β2-microglobulin, luteinizing hormone, human transferrin, and immunoglobulin E, were covalently attached by amine coupling to the dextran layer and the antigen binding capacities were evaluated for different amounts of bound antibody. High immunoreactivities were obtained with molar activities ranging from 0.6 to 1.5 over a wide range of surface concentrations. Interaction studies between carboxymethylated dextran surfaces and antibodies showed that, in comparison with unmodified gold surfaces, the modified surface had low nonspecific adsorption and high capacity for antibody immobilization.
The novel immobilization technique using a poly(amino acid) multilayer was designed for the SPR sensing platform. These poly(amino acid) multilayer was confirmed as a relatively hydrophilic surface with little tendency for nonspecific adsorption of biomolecules and a flat surface that the biomolecular interactions can occur uniformly on the sensing surface. The poly(amino acid) multilayer functionalized with biotin was investigated to control the surface density and to estimate the optimum space between ligands for effective SPR sensing. This multilayer study demonstrates the importance of surface density which is significantly sensitive for fabrication of surface functional group in the monitoring of kinetic phenomena.
Since the first application of the surface plasmon resonance (SPR) phenomenon for sensing almost two decades ago, this method has made great strides both in terms of instrumentation development and applications. SPR sensor technology has been commercialized and SPR biosensors have become a central tool for characterizing and quantifying biomolecular interactions. This paper attempts to review the major developments in SPR technology. Main application areas are outlined and examples of applications of SPR sensor technology are presented. Future prospects of SPR sensor technology are discussed.
Reliable, direct "sample-to-answer" capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s-->pi(*) transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein adsorption, facilitating detection of small DNA target sequences from these undiluted, unpurified complex biological mixtures unachievable with previously reported, analogous ssDNA/11-mercapto-1-undecanol monolayer surfaces [P. Gong, C.-Y. Lee, L. J. Gamble, D. G. Castner, and D. W. Grainger, Anal. Chem. 78, 3326 (2006)].
Poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes are extremely protein resistant polymer coatings that can reduce nonspecific adsorption of proteins from complex mixtures such as blood, sera and plasma. These coatings can be prepared via atom transfer radical polymerization with excellent control of their thickness and grafting density. We studied their direct functionalization with streptavidin and developed an assay for determining which coupling conditions afford the highest streptavidin loading efficiency. Disuccinimidyl carbonate was found to be the most efficient activating agent for covalent capture of the receptor. Using infrared and X-ray photoelectron spectroscopy, fluorescence microscopy, surface plasmon resonance, and ellipsometry, we examined how structural parameters such as the length of the oligo(ethylene glycol) side chain affect streptavidin functionalization, but also immobilization of biotinylated antibodies, subsequent selective secondary recognition and nonspecific binding of proteins. We found evidence that large macromolecules cannot infiltrate dense polymer brushes and that bulky antibody recognition occurs in the upper part of these coatings.
This work demonstrated the ultra-low fouling natural peptides composed of certain negatively and positively charged residues such as glutamic acid (E) or aspartic acid (D) and lysine (K), in the form of either alternating or randomly mixed charge. These peptide-based materials are major candidates as biodegradable nonfouling materials since their final metabolized products are natural amino acids. Although hydrophilic materials can generally reduce nonspecific binding to a certain extent, it is very challenging to achieve ultra-low fouling, which is critical for many biomedical applications, such as medical implants, drug delivery carriers, and biosensors. Based on the design principle of uniformly mixed charges and the selection of appropriate amino acid residues, the natural peptides developed exhibit high resistance to nonspecific protein adsorption (<0.3 ng/cm(2) adsorbed proteins) comparable to what is achieved by poly(ethylene glycol) (PEG)-based materials. Mixed charged groups, when uniformly distributed at the molecular level, can achieve ultra-low fouling properties similar to zwitterionic groups due to their strong hydration ability.
Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/mL, respectively. Minimizing these nonspecific interactions has allowed rapid and direct quantification of beta-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequently form a monolayer on gold in the form of 3-MPA-(AA)(n)-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-(AA)(n)-OH showed that the lowest nonspecific adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-(Ser)(5)-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from beta-lactamase by approximately 1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar beta-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media.
A new surface chemistry presenting an abundance of functional groups for ligand immobilization in an ultra-low fouling background all in one material for nanoparticles was introduced. This surface platform, as demonstrated by zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) coated nanoparticles, is not only ultra stable in undiluted human blood serum, but also can be conjugated to biomolecules conveniently and effectively. Thus, this surface chemistry is ideal to create multi-functional nanoparticles for targeted delivery and diagnostics. In addition, this work clearly shows that 10% blood serum commonly used to evaluate the stability of nanoparticles is insufficient and a new evaluation criterion with undiluted blood serum is recommended.
Human blood serum and plasma pose significant challenges to blood-contacting devices and implanted materials because of their high nonspecific adsorption onto surfaces. In this work, we investigated nonspecific protein adsorption from single protein solutions and complex media such as undiluted human blood serum and plasma onto poly(carboxybetaine acrylamide) (polyCBAA)-grafted surfaces at different temperatures. The polyCBAA grafting was done via atom-transfer radical polymerization (ATRP) with varying film thicknesses. The objective is to create a surface that experiences "zero" protein adsorption from complex undiluted human blood serum and plasma. Results show that protein adsorption from undiluted human blood serum, plasma, and aged serum on the polyCBAA-grafted surface is undetectable at both 25 and 37 degrees C by a surface plasmon resonance (SPR) sensor. This was achieved with a film thickness of approximately 21 nm. Furthermore, it is demonstrated that the polyCBAA surfaces after antibody immobilization maintain undetectable protein adsorption from undiluted human blood serum. This is the first time that an effective nonfouling material suitable for applications in complex blood media has been demonstrated.
Several risk factors for varicose veins have been identified: female gender, combined with obesity and pregnancy, occupations requiring standing for long periods, sedentary lifestyle, history of deep-vein thrombosis and family history. However, no specific gene variants related to a wide prevalence of varicosities in general population have been identified. Extracellular matrix composition, predominantly maintained by matrix metalloproteinases (MMPs), may affect the vein-wall structure, which may lead to dilation of vessels and cause varicosities.
MMP-1 (tissue collagenase I) and MMP-3 (stromelysin I) expression was found to be raised in varicose veins compared with normal vessels. Therefore, a study was conducted to evaluate a potential association between MMP1 and MMP3 promoter polymorphisms and a risk of varicose veins.
Genotyping for the presence of the polymorphisms -1607dupG (rs1799750) in MMP1 and -1171dupA (rs3025058) in the MMP3 promoter region was performed using PCR and restriction-fragment length polymorphism assays in a group of 109 patients diagnosed with varicose veins and 112 healthy controls.
The frequencies of the MMP1 and MMP3 alleles (minor allele frequency 0.440 in patients vs. 0.451 in the controls for MMP1-1607*G and 0.514 vs. 0.469 for MMP3-1171*dupA, respectively) and of genotypes did not differ significantly between patients and controls.
The MMP1-1607dupG and MMP3-1171dupA promoter polymorphisms are not valuable markers of susceptibility for varicose veins.
Self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG)-tethered molecules on gold are important for various biorelevant applications ranging from biomaterials to bioanalytical devices, where surface resistance to nonspecific protein adsorption is needed. Incorporation of a stimuli-responsive character to the OEG SAMs enables the creation of nonfouling surfaces with switchable functionality. Here we present an OEG-derived structure that is highly responsive to temperature changes in the vicinity of the physiological temperature, 37 degrees C. The temperature-responsive solution behavior of this new compound was demonstrated by UV-vis and nuclear magnetic resonance spectroscopy. Its chemisorption onto gold(111), and the retention of responsive behavior after chemisorption have been demonstrated by surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and atomic force and scanning tunneling microscopy. The OEG-derived SAMs have been shown to reversibly switch the wettability of the surface, as determined by contact angle measurements. More importantly, SPR and AFM studies showed that the OEG SAMs can be utilized to control the affinity binding of streptavidin to the biotin-tethered surface in a temperature-dependent manner while still offering the nonspecific protein-resistance to the surface.
In this contribution, we established a sandwich immunoassay system with a common spectrofluorometer to collect the plasmon resonance scattering (PRS) signals from silver nanoparticles (AgNPs) immunotargeted on glass slides. By taking the immunoreactions of goat antihuman IgG (Fc fragment specific) antibody (GAH-IgG), human immunoglobulin (H-IgG), and rabbit antihuman IgG (Fab fragment specific) antibody (RAH-IgG) as an example, we found that if a primary antibody (GAH-IgG) was first immobilized on the surface of glass slides and applied to capture target antigen (H-IgG), AgNPs-labeled secondary antibody (RAH-IgG) could be employed to detect the target antigen (H-IgG) by forming a sandwich immune complex on the surface of the glass slide. It was found that the PRS signals resulting from the AgNPs immunotargeted on the glass slides could be applied to the quantitative detection of H-IgG target antigen in the range of 10-1000 ng/mL with the limit of determination of 1.46 ng/mL (3sigma) under optimal conditions, which is sensitive and comparable with reported chemiluminescence immunoassays. With a dark-field microscope coupled with a spectral system, we measured the PRS features of single AgNPs immunotargeted on the glass slides, showing that the PRS of single nanoparticles might have potential applications in analytical chemistry. Further findings showed that the strong PRS signals from the AgNPs immunotargeted on the glass slides can be clearly seen and distinguished by naked eyes under the excitation of a common white light-emitting diode (LED) torch. Therefore, a visual PRS immunoassay system can be established easily with common glass slides and an LED torch.
Wavelength interrogation surface plasmon resonance (SPR) spectroscopy using a dove prism combines a simple and inexpensive optical design with high-resolution refractive index monitoring and biosensing. A BK7 dove prism inverts an optical image with a total internal reflection angle of 72.8 degrees , an angle active in SPR. Hence, a unique system can accomplish SPR biosensing using wavelength interrogation and also perform SPR imaging. This optical configuration advantageously uses a single axis optical path between each optical component, simplifying the optical design of SPR instruments without compromise of the analytical performance. Fluidics were also incorporated to the instrument design for efficient sample delivery. The SPR instrument is characterized in terms of refractive index (RI) sensitivity, RI resolution, reproducibility, and application for monitoring low concentration biological events. Data analysis methodologies are compared for improved resolution of the measured response. Raw data analyzed using a minimum hunting procedure results in RI resolution in the 10(-6) range, while pre-treating data with singular value decomposition improves the resolution by one order of magnitude. Depending on the spectrophotometer employed, the RI range accessible can be easily tuned; examples with a 550-850 nm and a 550-1100 nm spectrophotometers are shown and results respectively in RI ranges of 1.32-1.39 RIU and 1.32-1.42 RIU. Monitoring of microM concentration of beta-lactamase is performed using the wavelength interrogation configuration of the biosensor. Finally, a SPR image of a surface with a water droplet (volume=500 nL) was obtained using the dove prism SPR with a band pass filter and a CCD camera. SPR using a dove prism configuration combines advantages of portable SPR instruments, SPR imagers and research-grade SPR instruments in a unique platform.
Recent studies have shown that a loss of methylthioadenosine phosphorylase (MTAP) gene expression exerts a tumor-promoting effect, including induction of invasiveness, enhanced cell proliferation, and resistance against cytokines. To date, the molecular mechanisms underlying these effects remain unknown. Since the loss of MTAP expression resulted in induced secretion of 5'-deoxy-5'-(methylthio)adenosine (MTA), we hypothesized that MTA might modulate the observed effects. We first determined MTA levels produced by tumor cells in vitro and in situ by means of stable isotope dilution liquid chromatography tandem mass spectrometry. Subsequently, we revealed induction of matrix metalloproteinase (MMP) and growth factor gene expression in melanoma cells accompanied by enhanced invasion and vasculogenic mimicry. In addition, MTA induced the secretion of basis fibroblast growth factor (bFGF) and MMP3 from fibroblasts and the upregulation of activator protein-1 (AP-1) activity in melanoma cells and fibroblasts. In summary, we demonstrated a tumor-supporting role of MTA.
In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for protein immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct protein immobilization in two simple steps: surface activation and protein immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6h with <7ng/cm(2) of adsorbed proteins. The biological activity of the immobilized proteins was demonstrated with the detection of a model protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific protein adsorption to these surfaces.