No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Exploring the Versatility of Dendrimer-Stabilized Silver Nanoparticles Platforms: Synthesis, Characterization, and Protein Immobilization for Enhanced Biosensing Applications
Abstract
Surface Plasmon Resonance (SPR)-based biosensors have gained increasing prominence due to their ability to provide fast, accurate and real-time results. The functionalization of the SPR sensor chip is essential to...
The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth‐related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question.
We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C‐terminal short variable region (SVR) and inhibits its own translation.
The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. We propose that this negative protein‐transcript feedback loop may establish an on‐demand chaperone pool that allows for a rapid response to stress.
In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a noncell‐autonomous manner, potentially maintaining chaperone homeostasis between tissues.
Dendrimers, by virtue of their unique well-defined dendritic structure and multivalent cooperativity, hold great promise for a wide variety of applications, ranging from healthcare to energy production and environmental sustainability. However, dendrimer synthesis suffers from two inherent problems which greatly handicap their development and limit their application: the formation of structural defects caused by incomplete or side reactions, and difficulties associated with purification. Solid-phase synthesis may overcome both these problems, firstly by enabling complete chemical reactions through the use of a large excess of reagents, secondly by facilitating purification through simple washing and filtering steps. The end result is the speeded up reactions producing the desired product in high yield, with simultaneous suppression of by-products. In this review, we present the challenges and current state of research in solid-phase dendrimer synthesis, and provide our perspectives on its future development. We start with a short introduction to solid-phase synthesis and specific considerations for dendrimer construction. We then present exemplary studies to highlight the potential of, and challenges faced by, solid-phase dendrimer synthesis. Finally, we describe efforts to deliver more effective and reliable methods that will transform the synthesis of dendrimers and permit exploitation of their full potential.
Currently, several biosensors are reported to confirm the absence/presence of an abnormal level of specific human biomarkers in research laboratories. Unfortunately, public marketing and/or pharmacy accessibility are not yet possible for many bodily fluid biomarkers. The questions are numerous, starting from the preparation of the substrates, the wet/dry form of recognizing the (bio)ligands, the exposure time, and the choice of the running buffers. In this context, for the first time, the present overview summarizes the pre-functionalization of standard and nanostructured solid/flexible supports with cysteamine (Cys) and glutaraldehyde (GA) chemicals for robust protein immobilization and detection of biomarkers in body fluids (serum, saliva, and urine) using three transductions: piezoelectrical, electrochemical, and optical, respectively. Thus, the reader can easily access and compare step-by-step conjugate protocols published over the past 10 years. In conclusion, Cys/GA chemistry seems widely used for electrochemical sensing applications with different types of recorded signals, either current, potential, or impedance. On the other hand, piezoelectric detection via quartz crystal microbalance (QCM) and optical detection by surface plasmon resonance (LSPR)/surface-enhanced Raman spectroscopy (SERS) are ultrasensitive platforms and very good candidates for the miniaturization of medical devices in the near future.
Background: Upregulation of Heath Shock Protein 70 (HSP70) chaperones supports cancer cell survival. Their high homology causes a challenge to differentiate them in experimental or prevention and treatment strategies. The objective of this investigation was to determine similarities and differences of Hsp70, hsc70, Grp78 and Mortalin members of the HSP70 family encoded by HSPA1, HSPA8, HSPA5 and HSPA9 genes, respectively. Methods: Literature reviews were conducted using HSPA1, HSPA5, HSPA8 and HSPA9 gene or protein names or synonyms combined with biological or cancer-relevant terms. Ingenuity Pathway Analysis was used to identify and compare profiles of proteins that directly bind individual chaperones and their associated pathways. TCGA data was probed to identify associations of hsc70 with cancer patient survival. ClinicalTrials.gov was used to identify HSP70 family studies. Results: The chaperones have similar protein folding functions. Their different cellular effects are determined by co-chaperones and client proteins combined with their intra- and extra-cellular localizations. Their upregulation is associated with worse patient prognosis in multiple cancers and can stimulate tumor immune responses or drug resistance. Their inhibition selectively kills cancer over healthy cells. Conclusions: Differences in Hsp70, hsc70, Grp78 and mortalin provide opportunities to calibrate HSP70 inhibitors for individual cancers and combination therapies.
The carboxy-terminus of Hsp70-interacting protein (CHIP) is a ubiquitin ligase and co-chaperone belonging to Ubox family that plays a crucial role in the maintenance of cellular homeostasis by switching the equilibrium of the folding-refolding mechanism towards the proteasomal or lysosomal degradation pathway. It links molecular chaperones viz. HSC70, HSP70 and HSP90 with ubiquitin proteasome system (UPS), acts as a quality control system. CHIP contains charged domain in between N-terminal TPR and C-terminal Ubox domain. TPR domain interacts with the aberrant client proteins via chaperones while Ubox domain facilitates the ubiquitin transfer to the client proteins for ubiquitination. Thus, CHIP is a classic molecule that executes ubiquitination for degradation of client proteins. Further, CHIP has been found to be indulged in cellular differentiation, proliferation, metastasis and tumorigenesis. Additionally, CHIP can play its dual role as a tumor suppressor as well as an oncogene in numerous malignancies, thus acts as a double agent. Here, in this review, we have reported almost all substrates of CHIP established till date and classified them according to the hallmarks of cancer. In addition, we discussed about its architectural alignment, tissue specific expression, sub-cellular localization, folding-refolding mechanisms of client proteins, E4 ligase activity, normal physiological roles, involvement in various diseases and tumor biology. Further, we aim to discuss its importance in HSP90 inhibitors mediated cancer therapy. Thus, this report concludes that CHIP may be a promising and worthy drug target towards pharmaceutical industry for drug development.
A biosensor is an integrated receptor-transducer device, which can convert a biological response into an electrical signal. The design and development of biosensors have taken a center stage for researchers or scientists in the recent decade owing to the wide range of biosensor applications, such as health care and disease diagnosis, environmental monitoring, water and food quality monitoring, and drug delivery. The main challenges involved in the biosensor progress are (i) the efficient capturing of biorecognition signals and the transformation of these signals into electrochemical, electrical, optical, gravimetric, or acoustic signals (transduction process), (ii) enhancing transducer performance i.e., increasing sensitivity, shorter response time, reproducibility, and low detection limits even to detect individual molecules, and (iii) miniaturization of the biosensing devices using micro-and nano-fabrication technologies. Those challenges can be met through the integration of sensing technology with nanomaterials, which range from zero- to three-dimensional, possessing a high surface-to-volume ratio, good conductivities, shock-bearing abilities, and color tunability. Nanomaterials (NMs) employed in the fabrication and nanobiosensors include nanoparticles (NPs) (high stability and high carrier capacity), nanowires (NWs) and nanorods (NRs) (capable of high detection sensitivity), carbon nanotubes (CNTs) (large surface area, high electrical and thermal conductivity), and quantum dots (QDs) (color tunability). Furthermore, these nanomaterials can themselves act as transduction elements. This review summarizes the evolution of biosensors, the types of biosensors based on their receptors, transducers, and modern approaches employed in biosensors using nanomaterials such as NPs (e.g., noble metal NPs and metal oxide NPs), NWs, NRs, CNTs, QDs, and dendrimers and their recent advancement in biosensing technology with the expansion of nanotechnology.
The effective life-time of new antimicrobials until the appearance of the first resistant strains is steadily decreasing, which discourages incentives for commercialization required for clinical translation and application. Therefore, development of new antimicrobials should not only focus on better and better killing of antimicrobial-resistant strains, but as a paradigm shift on developing antimicrobials that prevent induction of resistance. Heterofunctionalized, poly-(amido-amine) (PAMAM) dendrimers with amide-conjugated vancomycin (Van) and incorporated Ag nanoparticles (AgNP) showed a 6-7 log reduction in colony-forming-units of a vancomycin-resistant Staphylococcus aureus strain in vitro, while not inducing resistance in a vancomycin-susceptible strain. Healing of a superficial wound in mice infected with the vancomycin-resistant S. aureus was significantly faster and more effective by irrigation with low-dose, dual-conjugated Van-PAMAM-AgNP dendrimer suspension than by irrigation with vancomycin in solution or a PAMAM-AgNP dendrimer suspension. Herewith, dual-conjugation of vancomycin together with AgNPs in heterofunctionalized PAMAM dendrimers fulfills the need for new, prolonged life-time antimicrobials killing resistant pathogens without inducing resistance in susceptible strains. Important for clinical translation, this better use of antibiotics can be achieved with currently approved and clinically applied antibiotics, provided suitable for amide-conjugation.
In this work we report the results acquired from molecular dynamics simulations as well as the optimization of different generations of polyamidoamine dendrimer. The analysis data revealed synthesized dendrimer as a suitable nanostructured candidate suitable for neutral as well as charged molecule delivery due to the presence of both electrostatic potential and van der Waals forces. The methyl ester terminating groups of half-generation dendrimers with characteristic IR peaks for carbonyl at 1670.41 cm⁻¹ tends to shift to 1514.17 cm⁻¹ on conversion to amide group of full-generation dendrimer. The study includes the usage of detailed analysis, demonstrating how molecular dynamics affect the dendrimer complexation. The present investigations provide an unprecedented insight into the computational and experimental system that may be of general significance for the clinical application of dendrimers.
In the original publication of the article, Fig. 5 was incorrect. The correct Fig. 5 appears as below.
Heat shock cognate 71-kDa protein (HSC70), a constitutively expressed molecular chaperon within the heat shock protein 70 family, plays crucial roles in maintaining cellular environmental homeostasis through implicating in a wide variety of physiological processes, such as ATP metabolism, protein folding and transporting, antigen processing and presentation, endocytosis, and autophagy. Notably, HSC70 also participates in multiple non-communicable diseases and some pathogen-caused infectious diseases. It is known that virus is an obligatory intracellular parasite and heavily relies on host machineries to self-replication. Undoubtedly, HSC70 is a striking target manipulated by virus to ensure the successful propagation. In this review, we summarize the recent advances of the regulatory mechanisms of HSC70 during viral infections, which will be conducive to further study viral pathogenesis.
A large body of literature is available on wound healing in humans. Nonetheless, a standardized ex vivo wound model without disruption of the dermal compartment has not been put forward with compelling justification. Here, we present a novel wound model based on application of negative pressure and its effects for epidermal regeneration and immune cell behaviour. Importantly, the basement membrane remained intact after blister roof removal and keratinocytes were absent in the wounded area. Upon six days of culture, the wound was covered with one to three-cell thick K14+Ki67+ keratinocyte layers, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing epidermal differentiation markers (K10, filaggrin, DSG-1, CDSN). Investigations about immune cell-specific manners revealed more T cells in the blister roof epidermis compared to normal epidermis. We identified several cell populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating a dermal efflux upon negative pressure. Together, our model recapitulates the main features of epithelial wound regeneration, and can be applied for testing wound healing therapies and investigating underlying mechanisms.
Cubosomes, a product of nanobioengineering, are self-structured lipid nanoparticles that act like drug-loaded theranostic probes. Here, we describe a simple method for the preparation of combinatorial drug-loaded cubosomes with, proof-of-principle, therapeutic effect against cancer cells, along with diagnostic capabilities. Anticancer drugs cisplatin and paclitaxel were loaded in the cubosomes in combination. The cubosomes were coated with a layer of poly-Ɛ-lysine, which helped avoid the initial burst release of drug and allowed for a slow and sustained release for better efficacy. Cubosomes were imaged by transmission electron microscope, and their dispersion analyzed in vitro by differential scanning calorimetric and X-ray diffractogram studies. The microscopic images depicted spherical polyangular structures, which are easily distinguishable. The analyses revealed that the drug is uniformly dispersed all through the cubosomes. Further characterization was carried out by zeta-potential measurement, in vitro release, and entrapment efficiency studies. The in vitro studies established that the coating of cubosomes successfully reduced the burst release of drugs initially and confirmed a slow, sustained release over increased time. Comparative cytotoxicity of coated, uncoated, and blank cubosomes was evaluated, using human hepatoma HepG2 cell line, and the formulations were found to be entirely nontoxic, similar to the blank ones. The therapeutic efficiency of the cubosomes against HeLa cells was confirmed by the impedance measurement and fluorescent imaging. Furthermore, the reduction in impedance in cells treated with coated combinatorial cubosomes proved the impairment of HeLa cells, as confirmed by fluorescence microscopy.
Contamination through Hg²⁺ ions in natural spring waters leads to a major destruction in human health and the universal ecosystem. Therefore, development of rapid, simple and cost‐effective methods for the specific and high sensitive determination of Hg²⁺ is of great importance. Among these methods, silver nanoparticles (Ag NPs) are used for the effective colorimetric detection of metal ions facilitating from the changes in the surface plasmon resonance (SPR) band. In this study, we have synthesized dendrimer‐encapsulated Ag NPs (Ag DENPs) by using Tris(2‐aminoethyl)amine (TREN) cored and amine‐terminated generation‐4 poly(amido amine) dendrimer (PAMAM), T4.NH2, as a novel template. The synthesized Ag DENPs were characterized by UV‐Vis, XRD, TEM and further used for the colorimetric and quantitative determination of Hg²⁺ ions. The developed Ag DENP sensor was tested against common present cations, Co²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Ni²⁺ Zn²⁺, Ba²⁺, Ca²⁺, Al³⁺, in the environment and found to be specific only for Hg²⁺ ions. Furthermore, limit of detection (LOD) for Hg²⁺ was found to be 1.18 ppb (5.88 nM). Results revealed that the proposed method can be successfully used with a good sensitivity and selectivity to determine mercury (II) ions in various water samples.
Octa-(3-chloropropyl) octasilsesquioxane (SS) was functionalized with a PAMAM dendrimer (SP) and characterized by some spectroscopic techniques such as Fourier Transform Infrared, Nuclear Magnetic Resonance, X-ray diffraction, X-ray Dispersive Energy Spectroscopy, Scanning Electron Microscopy and cyclic voltammetry. As a fast application, after there the Ag+ was adsorbed by SP and in a second step, the resulting material was reacted with potassium hexacyanoferrate (III) (AgHSP). The cyclic voltammogram of the AgHSP showed two redox pairs with formal potentials (Eθ’) of Eθ’(I) = 0.26 V and Eθ’(II) = 0.71 V (v = 20 mV s−1, KNO3 1.00 mol L−1, pH 7.00) assigned to processes Ag0 / AgI and FeII (CN)6 / FeIII (CN)6, respectively. The analytical application of this new material (AgHSP) was tested in the rapid detection of acid ascorbic by strong electrocatalysis using graphite paste electrode (GPE). The limit of detection (LOD) obtained for this system was 7.034 × 10−5 mol L−1 with an excellent amperometric sensitivity of 0.0835 A / mol L−1.
Functionalization of nanoparticle with specific groups is one of the most straightforward strategies to induce structural stability and specific cell responses from collagen based biomaterials. The effect of functionalised nanoparticles on triple helical conformational changes in collagen has not been understood well. For understanding the role of functionalization on collagen conformation, gold nanoparticles (Au NPs) prepared through wet chemical methods and functionalized with organic molecules (F-AuNPs) such as self-assembled monolayer (SAM), (3-aminopropyl) triethoxysilane (APTES), Polysaccharides (pectin and chitosan) and Poly(amido amine) PAMAM dendrimer (G0), were characterised and their interaction with collagen was studied. Protein conformational changes assessed by circular dichroism spectroscopy (CD) reveals that triple helical conformation of collagen was retained in presence of functionalized gold nanoparticle. The biocompatibility of functionalized gold nanoparticle was analysed against keratinocytes (HaCaT) cell by using (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) MTT assay. The result suggested that dendrimer functionalized gold nanoparticle exhibited higher cell viability when compared to other molecules functionalized gold nanoparticles studied. Based on the outcome of this study it can be envisioned that dendrimer functionalized gold nanoparticle mediated collagen materials are highly suitable for tissue engineering and cosmetic application.
The goals of this work were (1) to synthesize composite nanostructures comprised of amorphous calcium phosphate (ACP) loaded with silver nanoparticles using a spray pyrolysis method and (2) to demonstrate their potential for use in dental adhesives. Release of silver ions from these nanostructures could provide antibacterial activity, while release of calcium and phosphate ions could promote tooth remineralization. Precursor solutions were prepared with varying silver concentrations corresponding to 5, 10, and 15 mol % of the calcium content, then sprayed into a furnace (550 ºC) as droplets with a mean diameter near 2 μm. Each droplet is converted into a single solid microsphere via rapid heating. The synthesized particles were collected using a polymeric filter installed at the end of the reaction zone. Different quantities (2, 5, and 10 wt. %) of the nanocomposite material were mixed with a commercially available dental adhesive (Single Bond, 3M ESPE) which was then polymerized into discs for incubation in a solution simulating cariogenic conditions. Release of silver, calcium and phosphorus ions into the solution was measured for 1 month. The nanostructures of ~10 nm silver nanoparticles embedded into 100 nm – 2 μm ACP particles demonstrated good dispersion in the adhesive resin blend, which in application would shield surrounding tissues from direct contact with silver. The composite nanoparticles provided a quick initial release of ions after which the concentration of calcium, phosphorous, and silver in the incubation solution remained constant or increased slightly. The dispersibility and ion release of the new nanostructures may offer potential for use in dental materials to achieve anti-bacterial and remineralization effects.
A drug delivery carrier G4–FA–PEG with PEG and FA modified on the periphery of G4.0 poly(amidoamine) (PAMAM) dendrimer was synthesized, and doxorubicin (DOX) was encapsulated in the interior. The in vitro cytotoxicity and cellular uptake in gliomas cells were both enhanced via a FR-mediated endocytosis pathway.
Surface plasmon resonance (SPR) is a label-free detection method which has emerged during the last two decades as a suitable and reliable platform in clinical analysis for biomolecular interactions. The technique makes it possible to measure interactions in real-time with high sensitivity and without the need of labels. This review article discusses a wide range of applications in optical-based sensors using either surface plasmon resonance (SPR) or surface plasmon resonance imaging (SPRI). Here we summarize the principles, provide examples, and illustrate the utility of SPR and SPRI through example applications from the biomedical, proteomics, genomics and bioengineering fields. In addition, SPR signal amplification strategies and surface functionalization are covered in the review.
We describe a strategy for the determination of microRNA-126 (miRNA-126) that is based on the use of a glassy carbon electrode modified with (a) carboxy-terminated generation 3.5 poly(amidoamine) (PAMAM) dendrimer; (b) gold and silver nanoclusters, and (c) a chitosan-graphen composite. A peptide nucleic acid (PNA) was immobilized on the surface and serves as a receptor to hybridize miRNA-126. Subsequently, a digoxin labeled signal DNA is specifically recognized by the PNA. The analytical signal was finally generated by adding anti-digoxin antibody labeled with horse radish peroxidase. The biosensor was characterized by differential pulse voltammetry. A linear current-concentration relationship is found for miRNA-126 at a working voltage of 208 mV in the 1.0 fM to 10 nM concentration range. The limit of detection is 0.79 fM (at an S/N of 3). This biosensor displays good reproducibility, stability and selectivity.
Figure
A feasible strategy for microRNA-126 (miRNA-126) direct detection in the urine sample was described based on the synthesized carboxyl-terminated generation 3.5 (G 3.5) poly (amidoamine) (PAMAM) dendrimer with gold and silver alloy and chitosan-graphene sheets. Schematic illustrations of the electrochemical miRNA biosensor. (a) Synthesis of PAMAM-Au-Ag nanocomposite; (b) Assembly of the biosensor
Aluminum (Al) is recognized as a neurotoxin. Studies have confirmed that the neurotoxicity induced by Al may be related to tau hyperphosphorylation. Phosphorylated tau is degraded through the ubiquitin-proteasome pathway (UPP), in which the carboxyl terminus of Hsc70-interacting protein (CHIP) plays an important role. However, whether the CHIP plays a role in regulating tau hyperphosphorylation induced by Al is yet to be determined. The purpose of this study was to explore the molecular mechanism of the CHIP in tau hyperphosphorylation induced by AlCl3 in N2a cells. Mouse neuroblastoma cells (N2a) were exposed to different concentrations of AlCl3 (0, 0.5, 1, and 2mM) and treated with CHIP/CHIP shRNA/CHIP (ΔU-box)/CHIP (ΔTPR) plasmid transfection. The cell viability was determined by the CCK-8 kit. Protein expression was detected by Western blot. The interaction between CHIP and AlCl3 exposure on the proteins was analyzed by factorial design ANOVA. The results showed that Al can cause tau hyperphosphorylation, mainly affecting the pThr231, pSer262, and pSer396 sites of tau in N2a cells. UPP is involved in the degradation of tau hyperphosphorylation induced by Al in N2a cells, of which CHIP may be the main regulatory target. Both the U-box and TPR domains of CHIP are indispensable and play an important role in the regulation of tau hyperphosphorylation induced by AlCl3 in N2a cells.
Of the above 50 reported dendrimers families, the poly (amidoamine) (PAMAM) has remained one of the topmost explored classes due to its intended applications in potential areas ranging from nanomedicines to diagnostics, energy technologies, environmental remediation, analytical chemistry, and catalysis. The key features of PAMAM attractive to the scientific community are their controllable size in nanometers, globular three-dimensional branched architecture with a huge number of intramolecular cavities for host-guest interactions, facile synthesis methods, monodispersed nature, a substantial number of surface groups that can be manipulated chemically for controlling the physicochemical features and producing versatile products, low toxicity and cost, biocompatibility and easy fabrication to different substrates. In this paper, significant knowledge of PAMAM dendrimers is reviewed from the perspective of different structural parts, particularly the central core and the peripheral end groups. Additionally, the immobilization of PAMAM to various substrates such as metal nanoparticles (MNPs), carbonaceous nanomaterials, porous organic polymers, synthetic membranes, and electrodes has been presented. Besides this, the last part of the review is devoted to the applications of PAMAM dendrimers in analytical chemistry. Finally, the future outlook and perspective are addressed briefly.
Dendrimers are synthetic nano-molecules with spherical structure and a stardust topology. They have an amphiphilic nature and a hollow structure, with different layers over their central core structure representing each generation. Numerous drugs can be incorporated into the cavity present which helps solve a variety of problems experienced in the delivery of drugs such as poor solubility. Nowadays, dendrimers are being employed for chemotherapeutic drug delivery. The most common cancer worldwide is lung cancer which is caused due to various reasons, the major one being smoking. Dendrimers are presently utilised for the delivery of drugs to various targets and are proved to be successful in treating cancer, with their efficiency to treat lung cancer at various stages is being assessed. Although many nanoparticles (NPs) are available for their medical applications, from diagnosis to cancer treatment, dendrimers succeed amongst all due to most potential in terms of their structure, ability to provide good bioavailability and better stability than other NPs. There are mainly-three types of dendrimers used in treating lung cancer- PAMAM, PLL and PPI dendrimers. This review aims to describe the various properties of the dendrimers, investigate the properties of their various types and discover the reasons for their employment in treatment of lung cancer, the novelty being brought in them and the alterations which make them a potential chemotherapeutic drug delivery system.
The necessity for the development of highly sensitive and selective detection techniques for the investigation of biomolecules are inevitable. Surface plasmon resonance (SPR) based biosensor has developed as a sustainable and commercially applicable tool for this investigation. The detection of biomolecules, that are either toxic or useful to the environment, and the early diagnosis of disease biomarkers together come up with the key for better living. The exploration of this should give the prime concern for the real-time detection techniques that are cost-effective and least time-consuming. Here, we have briefly introduced the properties, classifications and characteristics of nanomaterials and that of the biosensors, we carefully analysed the properties of nanomaterials that can enhance sensor’s activity. We have given much importance to the recent development in the field of biosensing and the role of different nanomaterials including gold, silver, magnetic and 2D nanomaterials in enhancing the properties of SPR sensors under various configurations with explaining the principle and mechanism of SPR in brief. Along with this, we have addressed the phase matching schemes for SPR and the coupling of SPR with other modes such as guided modes, phonon polariton and defect modes in enhancing performance. The role of other plasmons such as Tamm plasmons and Bloch surface waves are also included in the review.
Research on paper substrates prepared by inkjet deposition of metal nanoparticles for sensing applications has become a hot topic in recent years; however, the design of such substrates based on the deposition of alloy nanoparticles remains less explored. Herein, we report for the first time the inkjet printing of dendrimer–stabilized colloidal metal nanoalloys for the preparation of paper substrates for Surface-enhanced Raman scattering (SERS) spectroscopy. To this end, nanoassemblies containing variable molar ratios of Au:Ag were prepared in the presence of poly(amidoamine) dendrimer (PAMAM), resulting in plasmonic properties that depend on the chemical composition of the final materials. The dendrimer–stabilized Au:Ag:PAMAM colloids exhibit high colloidal stability, making them suitable for the preparation of inks for long–term use in inkjet printing of paper substrates. Moreover, the pre-treatment of paper with a polystyrene (PS) aqueous emulsion resulted in hydrophobic substrates with improved SERS sensitivity, as illustrated in the analytical detection of tetramethylthiuram disulfide (thiram pesticide) dissolved in aqueous solutions. We suggest that the interactions established between the two polymers (PAMAM and PS) in an interface region over the cellulosic fibres, resulted in more exposed metallic surfaces for the adsorption of the analyte molecules. The resulting hydrophobic substrates show long–term plasmonic stability with high SERS signal retention for at least ninety days.
Dendrimers are well-defined, highly branched, multivalent and monodisperse molecules which host a range of attractive, yet functional, chemical and biological characteristics. A dendrimers accessible surface groups enable coupling to different functional moieties (e.g., antibodies, peptides, proteins, etc), which is further assisted by the dendrimers tailored size and surface charge. This adaptability allows for the preparation of molecularly precise vaccines with highly specific and predictable properties, and in conjunction with a dendrimers immune stimulating (adjuvanting) property, makes dendrimers attractive substrates for biomedical applications, including vaccines. This review highlights the structural and synthetic evolution of dendrimers throughout history, detailing the dendrimers role as both an adjuvant and carrier system for vaccine antigens, in addition to reviewing the development of commercially available vaccines for use in humans.
Excessive uric acid levels in the human body (hyperuricemia) are the main causes of kidney stones and diabetes. In this study, a layer-by-layer arrangement of polymers and nanocomposites is used as a new electrode sensing material for rapid and direct electrochemical determination of uric acid (UA). The electrode surface architecture was constructed by the incorporation of poly (amidoamine) dendrimer with 0.5 generation (poly (amidoamine) [PAMAM] [D-G0.5]) of multiwalled carbon nanotube-silver nanoparticles (MWCNT-AgNP) and a poly (neutral-red) (poly [NR]) polymer. The PAMAM (D-G0.5)/MWCNT-AgNP/poly (NR)-coated electrode has a good electrocatalytic activity for the determination of UA using cyclic voltammetry and showed remarkable enhancement in current response at a low-oxidation potential (0.3 V). Under optimal conditions, the developed electrochemical sensor showed an excellent and wide linear range for the determination of UA (i.e. 0.016 μM–2500 μM), and the limit of detection was found to be 0.005 μM. The modified sensor system demonstrated excellent sensitivity and selectivity toward the detection of UA in the presence of interfering substances, which are commonly found in urine and human fluid samples. Furthermore, the developed sensor has represented both reproducibility and excellent stability for the UA determination in real samples (human urine).
Drug delivery systems or vectors are usually needed to improve the bioavailability and effectiveness of a drug through improving its pharmacokinetics/pharmacodynamics at an organ, tissue or cellular level. However, emerging technologies with sensitive readouts as well as a greater understanding of physiological/biological systems have revealed that polymeric drug delivery systems are not biologically inert but can have innate or intrinsic biological actions. In this article, we review the emerging multiple innate biological/toxicological properties of naked polyamidoamine (PAMAM) dendrimer delivery systems in the absence of any drug cargo and discuss their correlation with the defined physicochemical properties of PAMAMs in terms of molecular size (generation), architecture, surface charge and chemistry. Further, we assess whether any of the reported intrinsic biological actions of PAMAMs such as their antimicrobial activity or their ability to sequester glucose and modulate key protein interactions or cell signaling pathways, can be exploited clinically such as in the treatment of diabetes and its complications.
The direction of this review is concentrating on the development of poly(amidoamine) (PAMAM) dendrimer encapsulated metal nanoparticles (NPs), so-called Dend-NPs as catalysts in the field of electrochemical energy conversion and storage. To date, Dend-NPs have been pointed out as well-organized and effective catalysts in the most popular electrochemical technologies, such as fuel cells, batteries, supercapacitors and water electrolyzers. Different strategies and technological progresses targeting efficient power and energy productions with excellent durability and performance retentions in such systems are emphasized. Dend-NPs action in electrochemical energy systems thrived by cathodic catalytic reactions, particularly, hydrogen evolution reaction (HER) in electrolyzers and oxygen reduction reaction (ORR) in fuel cells, and electrochemical (Faradaic and non-Faradaic) processes in batteries and supercapacitors are summarized. The metal NPs produced within the dendrimer by various approaches, conditions for obtaining zero-valent metals and synthesis methods for different types of metal NPs are discussed. Advancing knowledge of Dend-NPs synthesis, difficulties and their solutions and the best practice recommended for readers are explained in brief. Besides, stability, role of dendrimer, generation effects, terminal group effects and mechanisms to encapsulate each transition and precious metal NPs such as copper (Cu), nickel (Ni), platinum (Pt), palladium (Pd), silver (Ag), gold (Au) and bimetallic within the cavity of PAMAM dendrimers are highlighted. Moreover, PAMAM dendrimer and its generation are classified bearing in mind their viable use in energy technologies and applications where possible.
Bacterial colonization on implanted biomaterials remains a clinically significant problem. In order to achieve relatively long-term antibacterial activity and reduce the incidence of infections associated with the use of biomaterials, a salivary statherin protein (SSP) inspired poly(amidoamine) dendrimer (SSP-PAMAM-NH 2) was synthesized and characterized. PAMAM-NH 2 has numerous peripheral amino groups, and thus possesses effective antibacterial activity. The SSP bio-inspired peptide sequence DDDEEKC was conjugated to PAMAM-NH 2 since it has a strong capability of adsorbing on hydroxyapatite (HA). Moreover, SSP-PAMAM-NH 2 is a zwitterionic polymer possessing cationic amino groups and anionic carboxylic groups, thus it can form aggregates by intermolecular electrostatic interactions, thereby promoting its adsorption on HA. Adsorption tests by ATR-IR, UV, QCM-D, and CLSM, all indicated that SSP-PAMAM-NH 2 can tightly adsorb on the HA surface. We found that even after being incubated in PBS for 4 weeks, the SSP-PAMAM-NH 2 treated HA disks still retained stable antibacterial activity, while the inhibitory impact of PAMAM-NH 2 treated disks had disappeared. Animal experiments also demonstrated that SSP-PAMAM-NH 2 could significantly reduce infection of HA implanted into the medullary cavity of rats.
The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants.
We report a facile and versatile method to homogeneously deposit monolith membrane with uniform, high density of metallic nanoparticles via a “ship-in-a-bottle” strategy. Polyamidoamine (PAMAM) dendrimer, an excellent matrix for complexing with metal ions, is pre-infiltrated and applied as the directing agent for in-situ confined-formation of palladium nanoparticles (PdNPs) inside the mesopores. Efficiency of this method is demonstrated to prepare homogeneous PdNPs-deposited hierarchically porous graphitic carbon (HPGC) membrane with uniform metallic particle size (2.0-2.5 nm) and high palladium loading (∼34.4 wt%). Taking advantages of fast molecule diffusion rate in hierarchically porous structure and high conductivity of graphitic carbon substance, the PdNPs-dispersed HPGC membranes are applied as monolith electrodes for electrochemical applications. The PdNPs-deposited HPGC membrane electrode exhibits excellent electrocatalytic activity toward the catalytic oxidation of dopamine, uric acid and ascorbic acid, as well as high sensitivity and selectivity in simultaneous determination of these compounds in real serum samples. The limit of detections for dopamine, uric acid and ascorbic acid are 1.3×10⁻⁸, 2.6×10⁻⁸ and 3.7×10⁻⁸ M, respectively, at least one order lower than that achieved on electrochemical sensors reported previously. This work provides a versatile method for efficient preparation and stabilization of monodisperse metallic NPs in diverse porous materials, leading to possible applications in devices, catalysis, and electrochemical sensing.
Due to the small size, uniform dispersion and good stability, the dendrimer encapsulated nanoparticles (DENs) are widely used as nanocomposite materials. However, as a commonly used template, polyamidoamine (PAMAM) dendrimer is limited in the biological and medical fields because of its toxicity. Besides, nanoparticles are commonly encapsulated inside the dendrimer, which limits the effective contact with the reactants. In this paper, we presented a comprehensive study on the synthesis, characterization and application of 4-carbomethoxypyrrolidone modified polyamidoamine (PAMAM-Pyr) dendrimers-silver nanocomposites (Ag-PPDNCs). The silver nanoparticles were stabilized by PAMAM-Pyr, which combined the advantages of PAMAM and polyvinylpyrrolidone (PVP). Compared with particles prepared by PVP or PAMAM, silver nanoparticles prepared by PAMAM-Pyr had smaller particle size with narrow distribution of 4.78±0.57 nm, which was a kind of mixed type of DENs. In addition, Ag-PPDNCs showed very high activity in the reduction of 4-nitrophenol with the observed rate constant up to 7.2 × 10-3 s-1 and the turnover frequency was as high as 948.6 h-1, which was superior to other reported templates. These results demonstrated the significant role of PAMAM-Pyr in the preparation of Ag-PPDNCs with small particle size and excellent catalytic reduction property. Due to the good application performance and low toxicity, PAMAM-Pyr has great application potential in the fields of catalysis and medicine.
Design and development of electrochemical biosensors with improved selectivity, sensitivity and stability is one of the thrust areas of research in analytical and materials chemistry. In the present work, hemoglobin (Hb) was covalently immobilized on polyamidoamine (PAMAM) dendrimer encapsulated with gold nanoparticles (AuNPs), which was further utilized for the electrochemical detection of hydrogen peroxide (H2O2). Third generation PAMAM dendrimers were synthesized and AuNPs were encapsulated within the dendrimer network. Hb was covalently immobilized through glutaraldehyde cross-linking between the free amino groups of Hb and that of the PAMAM dendrimer. Hb/PAMAM-AuNPs was immobilized on a glassy carbon electrode (GCE) and the Hb/PAMAM-AuNPs/GCE modified electrode thus fabricated was characterized with electrochemical impedance spectroscopy and cyclic voltammetry. The Hb/PAMAM-AuNPs/GCE biosensor displayed well resolved redox peaks with anodic peak potential at −0.252 V and cathodic potential at −0.321 V, corresponding to Fe(III)/Fe(II) redox couple of heme active centre. Further, the developed Hb/PAMAM-AuNPs/GCE showed very good electrocatalytic activity for the reduction of H2O2 at a potential of −0.35 V. The Hb/PAMAM-AuNPs/GCE biosensor has shown impressive performance towards H2O2 determination in the concentration range from 20 μM to 950.22 μM. The sensitivity of the biosensor was calculated to be 35.07 μA μM⁻¹ cm⁻² with a detection limit of 6.1 μM. Also, the Hb/PAMAM-AuNPs/GCE modified electrode exhibited higher stability and good reproducibility.
Dense arrays of well-dispersed gold nanoparticles (AuNPs) on optical fibers are shown to bridge the gap in sensitivity and sensing performance between localized surface plasmon resonance (LSPR) and classical SPR sensing. A simple self-assembly method relying on a poly(styrene-b-4-vinylpyridine) (PS-b-P4VP) block copolymer brush layer was used to immobilize AuNPs of different diameters from 10 to 92 nm on optical fibers. In comparison with standard AuNP deposition methods using (3-aminopropyl)trimethoxysilane (APTMS) and polyelectrolytes, the sensitivity with the PS-b-P4VP templating method was found to be 3-fold better, a consequence of the smaller gap between particles and the presence of fewer AuNP aggregates. Hence, the sensitivity of the LSPR sensor for IgG was comparable to a classical SPR, also on optical fibers, and about 68% of that for a prism-based wavelength-interrogation SPR instrument. The reproducibility and the detection limit of the LSPR sensor were about the same as the SPR sensor. The enhanced performance of the LSPR sensors using the PS-b-P4VP block copolymer fabrication method paves the way for use of these LSPR biosensors in a smaller and more cost-effective platform.
Cardiovascular diseases, cancer, and diabetes are high mortality diseases, which account for almost two thirds of all deaths worldwide. Their early detection and continuous evaluation are fundamental for an improved patient prognosis and reduced socioeconomic impact. Current biosensor technologies are typically based on the analysis of whole blood samples from patients for the detection of disease-specific biomarkers. However, these technologies display serious shortcomings, such as reduced sensitivity and dynamic range, limited in vivo applicability, and lack of continuous monitoring. There is the urgent need for new diagnostic and treatment follow-up tools, which allow for the early detection of the pathology as well as for the continuous monitoring of the physiological responses to specific therapies. During the last years, a new generation of biosensor technologies with improved performance has emerged in the biomedical sector. The combination of advanced biomaterial methods, biochemical tools, and micro/nanotechnology approaches has resulted in the development of innovative three-dimensional (3D) biosensor platforms for advanced medical diagnosis. In this review, we report the most recent advances in the field of 3D biosensors for clinical applications, focusing on the diagnosis and monitoring of cardiovascular diseases, cancer, and diabetes. We discuss about their clinical performance compared to standard biosensor technologies, their implantable capability, and their integration into microfluidic devices to develop clinically-relevant models. Overall, we anticipate that 3D biosensors will drive us toward a new paradigm in medical diagnosis, resulting in real-time in vivo biosensors capable to significantly improve patient prognosis.
DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain. However, the interaction is transient, creating a challenge for detailed analysis. We investigated whether it would be possible to gain further understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale is to increase the proximity between the interacting partners to promote their natural interaction and facilitate the characterization of the interaction. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the full-length Hsp70 or in the SBD alone, for instance a higher protective effect against aggregation and being a monomer. Substrate binding also appear to exceed that of SBD alone as revealed by a decreased binding to bis-ANS, a probe for hydrophobic patches. This hypothesis is supported by the structural model created by small angle X-ray scattering, suggesting that the lid subdomain (SBDα) is partially opened in the J-SBD. Collectively, our results suggest a model in which J-domain binding may shift the Hsp70 equilibrium towards the monomer state, exposing hydrophobic
sites prone to substrate accommodation.
Since the first reports of Cu dendrimer encapsulated nanoparticles (DENs) published in 1998, the dendrimer-templating method has become the best and most versatile method for preparing ultrafine metallic and bimetallic nanoparticles (1-3 nm) with well-defined compositions, high catalytic activity and tunable selectivity. However, DENs have remained for the most part model systems with limited prospects for scale up and integration into high performance and reusable catalytic modules and systems for industrial-scale applications. Here, we describe a facile and scalable route to the preparation of catalytic polyvinylidene fluoride (PVDF) membranes with in-situ synthesized supramolecular dendrimer particles (SDPs) that can serve as hosts and containers for Pt(0) nanoparticles (2-3 nm). These new catalytic membranes were prepared using a reactive encapsulation process similar to that utilized to prepare Pt DENs by addition of a reducing agent (sodium borohydride) to aqueous complexes of Pt(II) + G4-OH/G6-OH polyamidoamine (PAMAM) dendrimers. However, the SDPs (2.4 μ average diameter) of our new mixed matrix PVDF-PAMAM membranes were synthesized in the dope dispersion without purification prior to film casting using (i) a low-generation PAMAM dendrimer (G1-NH2) as particle precursor and (ii) epichlorohydrin, an inexpensive functional reagent as crosslinker. In addition, the membrane PAMAM particles contain secondary amine groups (~1.9 meq per gram of dry membrane), which are more basic and thus have higher Pt binding affinity than the tertiary amine groups of the G4-OH and G6-OH PAMAM dendrimers. Proof-of-concept experiments show that our new PVDF-PAMAM-G1-Pt membranes can serve as highly active and reusable catalysts for the hydrogenation of alkenes and alkynes to the corresponding alkanes using (i) H2 at room temperature and a pressure of 1 bar and (ii) low catalyst loadings of ~1.4-1.6 mg of Pt. Using cyclohexene as model substrate, we observed near quantitative conversion to cyclohexane (~98%). The regeneration studies showed that our new Pt/membrane catalysts are stable and can be reused for five consecutive reaction cycles for a total duration of 120 hr including 60 hr of heating at 100oC under vacuum for substrate, product and solvent removal with no detectable loss of cyclohexene hydrogenation activity. The overall results of our study point to a promising, versatile and scalable path for the integration of catalytic membranes with in-situ synthesized SDP hosts for Pt(0) nanoparticles into high throughput modules and systems for heterogeneous catalytic hydrogenations, an important class of reactions that are widely utilized in industry to produce pharmaceuticals, agrochemicals and specialty chemicals.
The polyamidoamine dendrimer PAMAM was covalently bound to graphene oxide via amide formation between any peripheral amine group dendrimer and the carboxyl groups present in graphene oxide. This material (GOP) was characterized by different spectroscopic techniques, namely Raman Spectroscopy, X-Ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FT-IR) and X-Ray Diffraction (XRD). The hybrid material was shown to adsorb silver, that in turn complexes with hexacyanoferrate (III) ions (GOPAgH). The complexed hybrid material was electrochemically investigated using cyclic voltammetry. Some electroanalytical parameters, such as different supporting electrolytes, the supporting electrolyte concentrations, hydrogen ion concentration effects and scan rate were evaluated in order to obtain optimum responses. The GOPAgH material was successfully tested in the electrocatalytic detection of Isoniazid using a graphite paste electrode with Limits of Detection and Quantification of 5.0×10⁻⁶ mol L⁻¹ and 1.7×10⁻⁵ mol L⁻¹, respectively.
The synthesis, encapsulation and characterization of Au and Ag nanoparticles within silica nanospheres is described. Generation 4 amine-terminated PAMAM dendrimers were used as a template for both the nanoparticles and the silica nanosphere support synthesis. The synthesis of dendrimer-encapsulated (DENs) or stabilized (DSNs) Au or Ag nanoparticles was carried out first. The prepared DENs were characterized by UV–vis spectroscopy and TEM. The support precursor (silicic acid) is added to the preformed DENs/DSNs to form silica nanospheres for the fabrication of heterogeneous catalysts. In this case, the dendrimer is acting as a template for both the metallic nanoparticles and the support nanosphere. The dendrimer template was subsequently removed via calcination at 500 °C. These prepared catalysts were further characterized using various techniques such as TGA, BET, FTIR, PXRD, SEM, ICP-OES and EDX. The catalytic activity of these prepared catalysts was evaluated in the oxidation of benzyl alcohol. Preliminary catalytic evaluation was performed using two model reactions, namely, 4-nitrophenol reduction and oxidation of methylene blue. These types of catalysts have few advantages over other differently supported catalysts in that: (1) no agglomeration of nanoparticles that occurs during catalytic reaction even at elevated temperatures, (2) very little leaching or no leaching occurs during reaction, and (3) because the nanoparticles are not necessarily sitting on the support surface or within pores, relatively more active sites are available for reaction to take place.
A novel high performance liquid stationary phase was prepared by grafting poly amidoamine (PAMAM) dendrimers to poly (styrene-divinyl benzene-glycidylmethacrylate) matrix in this work. Different generations of PAMAM dendrimers grafted to polymer matrices containing different glycidylmethacrylate amounts by reacting with epoxy groups in glycidylmethacrylate molecules. The synthesized PAMAM dendrimers and stationary phase were characterized by fourier transform infrared spectroscopy, scanning electron microscope, N2 adsorption/desorption, elemental analysis and thermo gravimetric analyzer. The proposed stationary phase was suitable for rapid separation of phenylene diamine isomers and aminobenzene. The separation mechanism of benzene rings attraction and amino repulsion was verified by retention behaviors of methylamine, phenylamine, phenylene diamine, naphthylamine and toluene. The reliability of the stationary phase was demonstrated by separation and determination of phenylene diamine isomers including m-phenylene diamine, o-phenylene diamine and p-phenylene diamine in hair dye precursors within 10 min. Satisfactory resolution and short analysis time make the columns promising for determination of aniline compounds in various fields.
An electrochemical sensor for detection of paracetamol (PAT) was reported employing multiwalled carbon nanotubes (MWCNTs) and the fourth generation poly(amidoamine) dendrimers (G4.0 PAMAM). The strategy of this work lies on carboxylated MWCNTs and amine terminated G4.0 PAMAM form a stable covalent linkage via covalently layer-by-layer (LbL) self-assembly, which provide the larger surface areas for PAT adsorption efficiently. The constructed electrode was characterized first by fourier transform infrared spectroscopy and scanning electron microscope，and then the electrochemical behavior of PAT on (MWCNTs–G4.0)6/GCE was investigated in detail by cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry. Under the optimal conditions, the experimental results demonstrated that constructed sensor exhibited excellent catalytic activity toward PAT, and obtained a wide linear range from 3.0×10-7 – 2.0× 10-4 M with a limit of detection (LOD) of 1.0 × 10-7 M (S/N=3). Moreover, it could also be applied to the determination of PAT in commercial tablets and human serum, and the satisfactory results confirm the applicability of this sensor in practical analysis.
The catalytic oxidation of methylene blue was investigated in the presence of dendrimer generation five terminated NH2 encapsulated silver (AgDENs), and gold (AuDENs) nanoparticles as catalyst. The synthesis process of encapsulated metal nanoparticles were monitored by UV/vis spectrophotometry and their average diameters determined by transmission electron microscopy analysis, respectively. The kinetics was monitored using UV/vis spectrophotometry, under pseudo-first order conditions, resulting in observed rate constants kobs. Langmuir-Hinshelwood approach was applied to model the kinetic process. The adsorption of methylene blue on the catalyst surface is related to the type of metal nanoparticle present, and presents the rate-determining step.
Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small 'lid' (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents.
Enzyme immunoassay (EIA) has been the primary subject of a number of books and reviews, and an important concern of other recent books and reviews. Enzymes are the most versatile and popular class of labeling substances used for immunoassays, and they have an assured future. The invention of immunoenzymatic staining in the mid-1960s is said to have been the beginning of enzyme immunoassay (EIA), and this enabled the preparation of permanent photomicroscopic slides with visualization of specific antigens. In 1971, enzymes were introduced as alternatives to radioisotopes in immunoassays. Enzymes are perhaps the most varied class of labeling substances. About 1990, the most common enzyme label in new immunoassays was horseradish peroxidase, with alkaline phosphatase in second place. The term enzyme-mediated immunoassay encompasses, as well as all assays with enzymes as the labeling substance, assays with labels containing enzyme substrates, enzyme inhibitors, coenzymes, or enzyme cofactors. The operation of such assays depends on the use of one or more enzymes as ancillary reagents. Enzymes are also exploited in many other ways for the benefit of immunoassays. To prepare conjugates with enzyme, the method of conjugation used is particularly mild so as to retain both immuno- and enzymatic activities.
