Detection of the antiepileptic drug phenytoin using a single free-standing piezoresistive microcantilever for therapeutic drug monitoring

ArticleinBiosensors & Bioelectronics 59C:233-238 · March 2014with 200 Reads
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  • Article
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    Direct, small-molecule determination of the antiepileptic drug, valproic acid, was investigated by a label-free, nanomechanical biosensor. Valproic acid has long been used as an antiepileptic medication, which is administered through therapeutic drug monitoring and has a narrow therapeutic dosage range of 50–100 μg· mL −1 in blood or serum. Unlike labeled and clinically-used measurement techniques, the label-free, electrical detection microcantilever biosensor can be miniaturized and simplified for use in portable or hand-held point-of-care platforms or personal diagnostic tools. A micromachined microcantilever sensor was packaged into the micro-channel of a fluidic system. The measurement of the antiepileptic drug, valproic acid, in phosphate-buffered saline and serum used a single free-standing, piezoresistive microcantilever biosensor in a thermally-controlled system. The measured surface stresses showed a profile over a concentration range of 50–500 μg· mL −1 , which covered the clinically therapeutic range of 50–100 μg· mL −1 . The estimated limit of detection (LOD) was calculated to be 45 μg· mL −1 , and the binding affinity between the drug and the antibody was measured at around 90 ± 21 μg· mL −1 . Lastly, the results of the proposed device showed a similar profile in valproic acid drug detection with those of the clinically-used fluorescence polarization immunoassay.
  • Article
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    Foodborne pathogens, especially bacteria, are explicitly threatening public health worldwide. Biosensors represent advances in rapid diagnosis with high sensitivity and selectivity. However, multiplexed analysis and minimal pretreatment are still challenging. We fabricate a gold nanoparticle (Au NP)-amplified microcantilever array biosensor that is capable of determining ultralow concentrations of foodborne bacteria including Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Shigella, etc. The method is much faster than using conventional tools without germiculturing and PCR amplification. The six pairs of ssDNA probes (ssDNA1 + ssDNA2 partially complementary to the target gene) that originated from the sequence analysis of the specific gene of the bacteria were developed and validated. The ssDNA1 probes were modified with -S-(CH2)6 at the 5′-end and ready to immobilize on the self-assembled monolayers (SAMs) of the sensing cantilevers in the array and couple with Au NPs, while 6-mercapto-1-hexanol SAM modification was carried out on the reference cantilevers to eliminate the interferences by detecting the deflection from the environment induced by non-specific interactions. For multianalyte sensing, the target gene sequence was captured by the ssDNA2-Au NPs in the solution, and then the Au NPs-ssDNA2-target complex was hybridized with ssNDA1 fixed on the beam of the cantilever sensor, which results in a secondary cascade amplification effect. Integrated with the enrichment of the Au NP platform and the microcantilever array sensor detection, multiple bacteria could be rapidly and accurately determined as low as 1–9 cells/mL, and the working ranges were three to four orders of magnitude. There was virtually no cross-reaction among the various probes with different species. As described herein, it holds great potential for rapid, multiplexed, and ultrasensitive detection in food, environment, clinical, and communal samples.
  • Article
    As a highly sensitive nanomechanical sensor, microcantilever sensor is widely used in biochemical detection. Aflatoxin B1 (AFB1), a hepatocarcinogen widely present in food and food materials, is highly dangerous to human health, and new sensitive methods to detect AFB1 are needed. Here, we developed a microcantilever-array-based immunosensor used in stress mode to detect AFB1with the obvious advantages of a high sensitivity, rapidity, label-free, quantitative, and ability to be performed in real-time. The microcantilever was functionalized with a sulfhydrylated anti-AFB1 antibody, and an ELISA was used to validate the activity of the antibody on the microcantilever. Deflection of the microcantilever corresponding to different AFB1 concentrations was monitored in real-time. The detection limit of the microcantilever sensor in stress mode was lowered to 0.03 ng/mL for AFB1, which is a significant improvement in comparison with icELISA or a microcantilever sensor operated in dynamic mode. We also successfully detected AFB1 dissolved in a peanut solution. The microcantilever sensor in stress mode provides a new method for detecting extremely low concentrations of AFB1 and may have great potential for food quality control and public health protection.
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    Optimal dose selection in clinical trials is problematic when efficacious and toxic concentrations are close. A novel quantitative approach follows for optimizing dose titration in clinical trials. A system of pharmacokinetics (PK), pharmacodynamics, efficacy, and toxicity was simulated for scenarios characterized by varying degrees of different types of variability. Receiver operating characteristic (ROC) and clinical trial simulation (CTS) were used to optimize drug titration by maximizing efficacy/safety. The scenarios included were a low‐variability base scenario, and high residual (20%), inter‐occasion (20%), inter‐individual (40%), and residual plus inter‐individual variability scenarios, and finally a shallow toxicity slope scenario. The percentage of subjects having toxicity was reduced by 87.4% to 93.5%, and those having efficacy was increased by 52.7% to 243%. Inter‐individual PK variability may have less impact on optimal cut‐off values than other sources of variability. ROC/CTS methods for optimizing dose titration offer an individualized approach that leverages exposure‐response relationships. This article is protected by copyright. All rights reserved.
  • Article
    Recently, microcantilever-based technologies are playing more and more important roles in early diagnosis of cancer due to their high sensitivity, fast response, low cost, small reagent consumption, portability, real-time, labelfree detection, and so on. However, in conventional cantilever sensors working on mass-loading principle, the change of stiffness coefficient k is neglected. This results in distinct error for mass detection. Some researchers tried a local immobilization method to eliminate the undesired effect of k. But the change of k in this method still brings unexpected error. An accurate theoretical model is needed to take the effect of k change into account in the local immobilization approach. A micro-cavity was designed in the free end of the cantilever for local antibody immobilization in our work, thus the adsorption-induced variation of k can be dramatically reduced compared to that caused by adsorption of the whole lever. In addition, an analytical model has been established to eliminate the effect of adsorption-induced lever stiffness change and has been applied to precise mass detection of cancer biomarker AFP, the detected AFP antigen mass (7.6 pg/ml) is close to the calculated one (5.5 pg/ml), two orders of magnitude better than the value by the fully antibody-immobilized cantilever sensor. These approaches will promote clinical application of the cantilever sensors in early diagnosis of cancer.
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    Early diagnosis is a very fundamental issue in treating most diseases and for this purpose microcantilevers are very effective and reliable devices. In this work, four models of biosensor-based microcantilever are compared and a novel design with high sensitivity, quality factor, and accuracy is proposed. A truss structure is designed near the anchored end of the microcantilever, which improves the sensitivity in order to increase detection accuracy. A linear relationship between resonance frequency shift and masses has been estimated for all the designs. High quality factor, which increases the accuracy of measurement, is taken into account as the other benefit of the proposed design. These microcantilevers can be used as an array for multiple early diagnosis of cancer and moreover these features will improve clinical applications of cantilever sensors for early disease diagnosis.
  • Article
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    A novel microcantilever biosensor was batch-fabricated with IC compatible MEMS technology for joint detection of liver cancer biomarkers with high sensitivity, high throughput, high specification, and good precision. A microcavity was designed in the free end of the cantilever for local antibody-immobilization using micro printing system, which can dramatically reduce the effect of adsorption-induced k variation. A linear relationship between the resonance frequency shift and the antigen concentration was observed for three liver cancer biomarkers, AFP, GGT-2, and HGF. In addition, the presented immunosensing method has little cross-reactivity to different antigen, paving the way to a highly specific technique. These approaches will promote clinical application of the cantilever sensors in cancer early diagnosis.
  • Article
    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.
  • Article
    Therapeutic drug monitoring (TDM) is required for pharmaceutical drugs with dosage limitations or toxicity issues where patients undergoing treatment with these drugs require frequent monitoring. This allows for the concentration of such pharmaceutical drugs in a patient's biofluid to be closely monitored in order to assess the pharmacokinetics, which could result in an adjustment of dosage or in medical intervention if the situation becomes urgent. Biosensors are a class of analytical techniques competent in the rapid quantification of therapeutic drugs and recent developments in instrumental platforms and in sensing schemes, as well as the emergence of nanobiosensors, have greatly contributed to the principal examples of these sensors for therapeutic drug monitoring. Based on initial success stories, it is clear that (nano)biosensors could pave the way for therapeutic drug monitoring of many commonly administered drugs and for new drugs that will be introduced to the market allowing for safe and optimal dosing across a wide range of pharmaceuticals. In this review, we report on the recent developments in biosensing and nanobiosensing techniques and, focussing mainly on anti-cancer agents and antibiotics, we discuss the different classes of molecules upon which therapeutic drug monitoring has already been successfully applied. The potential contributions of (nano)biosensors are also reviewed for the emerging areas of therapeutic response monitoring, where markers are monitored to ensure compliance of a patient to a treatment and in the area of cellular response to therapeutic drugs in order to identify cytotoxic effects of drugs on cells or to identify patients responding to a drug.
  • Article
    A simple and efficient bioanalytical method for simultaneous determination of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), and its active metabolite carbamazepine 10,11-epoxide (CBZE) in human plasma using online solid phase extraction (SPE)-liquid chromatography (LC) coupled with high resolution mass spectrum (HRMS) under targeted MS/MS (t-MS2) analysis mode has been developed. The procedure integrated an automated sample clean-up of human plasma by Oasis®HLB SPE cartridge, a separation by ZORBAX SB-C18 analysis column, and a quantification by Q-Exactive Hybrid Quadrupole-Orbitrap. The total running time was 13 min. The lower limit of quantification (LLOQ) of PB, PHT, CBZ, and CBZE were 0.008, 0.008, 0.0016 and 0.0016 μg mL−1 respectively and the linearities were in the range of 0.008-2.500, 0.008-2.500, 0.0016-0.500 and 0.0016-0.500 μg mL−1 respectively. The mean recovery was between 91.82% and 108.27% and the matrix effect was between 93.29% and 102.09%. The relative standard deviations of interday and intraday were less than 6.41%. The method has been successfully applied in therapeutic drug monitoring (TDM) of four Chinese epilepsy patients. This fully automated, simple, sensitive and reliable online-SPE-LC-HRMS/MS method serves well for TDM of PB, PHT, CBZ and CBZE at clinics for either single or combination treatment.
  • Article
    A novel microcantilever biosensor was batch-fabricated with integrated circuit compatible micro-electromechanical system technology for joint detection of liver cancer biomarkers with high sensitivity, high throughput, high specification, and good precision. A micro-cavity was designed in the free end of the cantilever for local antibody-immobilization using micro-printing system, which can dramatically reduce the effect of adsorption-induced stiffness coefficient k variation. A linear relationship between the resonance frequency shift and the antigen concentration was observed for three liver cancer biomarkers, alpha-fetoprotein, gamma-glutamyl transpeptidase II, and hepatocyte growth factor. In addition, the presented immunosensing method has little cross reactivity to different antigens, paving the way to a highly specific technique. These approaches will promote clinical application of the cantilever sensors in early cancer diagnosis.
  • Article
    Piezoresistive micro/nano cantilever sensors have been extensively utilized in the detection and quantification of miniscule forces generated due to the physical interaction of biomolecules. Due to the ultra-sensitive nature of piezoresistive cantilever sensors, they have found numerous applications, especially in the development of point-of-care testing systems for healthcare applications. Over the years, silicon-on-insulator (SOI) wafers have been widely utilized to realize silicon based piezoresistive cantilever sensors due to their performance and fabrication related advantages. Treatise encompasses examples where researchers have retained the buried oxide (BOX) layer of SOI wafers to realize piezoresistive cantilever devices. In this paper, we investigate the impact of BOX layer of SOI wafers on the performance of composite piezoresistive cantilever biosensors. Typically, the response of piezoresistive cantilever sensors is evaluated by considering only the electro-mechanical response. However, the design of such sensors is a multi-variant problem due to (i) their composite structure and (ii) interdependent nature of the electrical, mechanical and thermal characteristics. Therefore, in the present study, we devise a new figure of merit (FoM) defined as the product of sensitivity ratio and the square of resonant frequency of the sensor. The sensitivity ratio represents the ratio of the electrical sensitivity due to surface stress and thermal sensitivity of the sensor. The proposed FoM takes into account the interplay between the electrical, mechanical and thermal response of the sensor. Finite element method based numerical simulations are performed to model and investigate the influence of BOX layer on the thermo-electro-mechanical response of a square shaped silicon piezoresistive cantilever sensor. Simulation results show that the presence of the supporting BOX layer enhances the mechanical stability i.e. spring constant and resonant frequency of the sensor. In addition, it is found that the BOX layer reduces the zero bias deflection of the cantilever due to difference in TCE of the constituent layer materials. Furthermore, it is shown that by dimensional optimization of the thicknesses of the constituent layers, sensitivity ratio of the sensor with BOX layer can be improved up to 2.96.
  • Article
    Early liver cancer diagnosis has clinical significance in treating cancer. Joint detection of multiple biomarkers has been considered as an effective and reliable method for early cancer diagnosis. In this work, a novel biosensor based on microcantilever array was batch-fabricated for multiple liver cancer biomarkers detection with high sensitivity, high accuracy, high throughput, and high specification. A micro-cavity was designed in the free end of the cantilever for local reaction between antibody and antigen, which can dramatically reduce the effect of adsorption-induced stiffness coefficient k variation. Furthermore,the pillar arrays in the micro-cavity were designed for increasing detection upper limit. A linear relationship between the relative frequency shift and the antigen concentration was observed for three liver cancer biomarkers, alpha-fetoprotein (AFP), γ-glutamyltranspeptidase II (GGT-2), and hepatocyte growth factor (HGF). Slight cross-reaction response to different antigens ensures high specificity of the sensor. These features will promote clinical application of the cantilever sensors in early cancer diagnosis.
  • Chapter
    Marketing specialists like to give new products exciting names and use comparisons that spark the imagination of the potential buyer. Many products have been compared to Star Trek technologies in name or description in the hopes that consumers will equate them with the space age items from the franchise. In most cases this is a stretch, but the XPrize Foundation currently is running a tricorder X Prize worth 10 million dollars. Teams must produce a small device to monitor vital signs and diagnose many diseases and conditions, all without the help of a medical professional. This is truly Star Trek-like. However it is incomplete. Star Trek tricorders are capable of collecting huge amounts of primary sensor data in many areas, including meteorology, biology, geology, geography, and materials sciences. To approximate these functions, smartphone applications and dongles are being developed that can sense all manner of environmental and physical data—even cancer, and then interpret the data for our use or collate it with other data on the Internet of Things.
  • Article
    We developed a microcantilever array biosensor instrument based on optical readout from a microcantilever array in fluid environment. The microcantilever signals were read out sequentially by laser beams emitted from eight optical fibers. The optical fibers were coupled to lasers, while the other ends of the fibers were embedded in eight V-grooves with 250 μm pitch microfabricated from a Si wafer. Aspherical lens was used to keep the distance between lasers. A programmable logic controller was used to make the system work stably. To make sure that the output of lasers was stable, a temperature controller was set up for each laser. When the deflection signal was collected, lasers used here were set to be on for at least 400 ms in each scanning cycle to get high signal-to-noise ratio deflection curves. A test was performed by changing the temperature of the liquid cell holding a microcantilever array to verify the consistent response of the instrument to the cantilever deflections. The stability and conformance of the instrument were demonstrated by quantitative detection of mercury ions in aqueous solution and comparison detection of clenbuterol by setting test and reference cantilevers. This microcantilever array detection instrument can be applied to highly sensitive detection of chemical and biological molecules in fluid environment.
  • Article
    Array sensing is increasingly important in the development of microcantilever (MC) sensors, and response consistency is the foundation for MC array sensing. In the present work, we investigated the response consistency of MC array sensing. The responses of two types of commercially available MC arrays were studied under conditions of temperature change, solution replacement and biochemical molecular interaction. For the thermal response, the deflections of both arrays were found to be proportional to temperature, and the responses of the MCs in both arrays were consistent with each other. The thermal response sensitivity for each MC during temperature increase and decrease also showed good consistency. Moreover, the MC array showed good consistency for the response induced by solution replacement. Finally, we also demonstrated that the MC array had good consistency in biochemical detection, exemplified by aflatoxin antibody-antigen binding. The good response consistency makes this technology reliable and accurate for biochemical sensing.
  • Article
    Full-text available
    In the last decade, microelectromechanical systems (MEMS) SU-8 polymeric cantilevers with piezoresistive readout combined with the advances in molecular recognition techniques have found versatile applications, especially in the field of chemical and biological sensing. Compared to conventional solid-state semiconductor-based piezoresistive cantilever sensors, SU-8 polymeric cantilevers have advantages in terms of better sensitivity along with reduced material and fabrication cost. In recent times, numerous researchers have investigated their potential as a sensing platform due to high performance-to-cost ratio of SU-8 polymer-based cantilever sensors. In this article, we critically review the design, fabrication, and performance aspects of surface stress-based piezoresistive SU-8 polymeric cantilever sensors. The evolution of surface stress-based piezoresistive cantilever sensors from solid-state semiconductor materials to polymers, especially SU-8 polymer, is discussed in detail. Theoretical principles of surface stress generation and their application in cantilever sensing technology are also devised. Variants of SU-8 polymeric cantilevers with different composition of materials in cantilever stacks are explained. Furthermore, the interdependence of the material selection, geometrical design parameters, and fabrication process of piezoresistive SU-8 polymeric cantilever sensors and their cumulative impact on the sensor response are also explained in detail. In addition to the design-, fabrication-, and performance-related factors, this article also describes various challenges in engineering SU-8 polymeric cantilevers as a universal sensing platform such as temperature and moisture vulnerability. This review article would serve as a guideline for researchers to understand specifics and functionality of surface stress-based piezoresistive SU-8 cantilever sensors. Open image in new window
  • Article
    In this work, we fabricated gold-nanowires (Au NWs) on flexible substrates by direct current electrochemical deposition. Then, we electropolymerized a polypyrrole layer onto the Au NWs via cyclic voltammetry (CV) as the molecular imprinted polymer biosensor to detect dopamine (DA). The influence of the scan rate of CV for the sensor preparation was optimized. CV and differential pulse voltammetry (DPV) were used to measure different DA concentrations. The Au/polypyrrole core/shell nanowires sensor showed good DPV signal intensity at 805 μA mM−1 and the determined linear range for DA was 4 × 10−7 to 1 × 10−5 M (N = 3).
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    Full-text available
    Since the late 1980s there have been spectacular developments in micromechanical or microelectro-mechanical MEMS systems which have enabled the exploration of transduction modes that involve mechanical energy and are based primarily on mechanical phenomena. As a result an innovative family of chemical and biological sensors has emerged. In this article, we discuss sensors with transducers in a form of cantilevers. While MEMS represents a diverse family of designs, devices with simple cantilever configurations are especially attractive as transducers for chemical and biological sensors. The review deals with four important aspects of cantilever transducers: i operation principles and models; ii microfabrication; iii figures of merit; and iv applications of cantilever sensors. We also provide a brief analysis of historical predecessors of the modern cantilever sensors. © 2004 American Institute of Physics.
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  • The similar tendency of increasing phenytoin drug concentrations in detection utilizing the traditional FPIA technique and the present piezoresistive microcantilever
    • Fig
    Fig. 5. The similar tendency of increasing phenytoin drug concentrations in detection utilizing the traditional FPIA technique and the present piezoresistive microcantilever.
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    This study demonstrates a novel method for electrical detection of C-reactive protein (CRP) as a means of identifying an infection in the body, or as a cardiovascular disease risk assay. The method uses a single free-standing, thermally controlled piezoresistive microcantilever biosensor. In a commonly used sensing arrangement of conventional dual cantilevers in the Wheatstone bridge circuit, reference and gold-coated sensing cantilevers that inherently have heterogeneous surface materials and different multilayer structures may yield independent responses to the liquid environmental changes of chemical substances, flow field and temperature, leading to unwanted signal disturbance for biosensing targets. In this study, the single free-standing microcantilever for biosensing applications is employed to resolve the dual-beam problem of individual responses in chemical solutions and, in a thermally controlled system, to maintain its sensor performance due to the sensitive temperature effect. With this type of single temperature-controlled microcantilever sensor, the electrical detection of various CRP concentrations from 1 µg/mL to 200 µg/mL was performed, which covers the clinically relevant range. Induced surface stresses were measured at between 0.25 N/m and 3.4 N/m with high reproducibility. Moreover, the binding affinity (KD) of CRP and anti-CRP interaction was found to be 18.83 ± 2.99 µg/mL, which agreed with results in previous reported studies. This biosensing technique thus proves valuable in detecting inflammation, and in cardiovascular disease risk assays.
  • Article
    A highly sensitive fluorometric high-performance liquid chromatographic method was developed for the simultaneous determination of phenytoin and its major metabolites [5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin]. After extracting these compounds and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) as an internal standard from serum (50 mu l) with ethyl acetate, they were further converted into the corresponding fluorescent derivatives by a reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and dibenzo-18-crown-6 in acetonitrile. The derivatives were separated by reversed-phase chromatography on a YMC-Pack ODS-A column with a mixture of acetonitrile-50 mM phosphate buffer (pH 7.0) (4:6, v/v) as a mobile phase, and were then detected spectrofluorometrically at 448 nm with excitation at 365 nm. The detection limits for phenytoin, 5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin added to serum were 0.6, 3.0 and 0.8 ng (2.4, 11 and 3.1 pmol) ml(-1) serum at a signal-to-noise ratio of three. The method was applied to determine the unbound- and total-phenytoin and the metabolites levels in the serum obtained from two healthy volunteers, after oral administration of the drug.
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    Surface stress changes and kinetics were measured in situ during the self-assembly of alkanethiols on gold by means of a micromechanical sensor. Self-assembly caused compressive surface stress that closely followed Langmuir-type adsorption kinetics up to monolayer coverage. The surface stress at monolayer coverage increased linearly with the length of the alkyl chain of the molecule. These observations were interpreted in terms of differences in surface potential. This highly sensitive sensor technique has a broad range of applicability to specific chemical and biological interactions.
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  • Article
    This study demonstrated a microcantilever biosensor for enhancement of capture antibody immobilization. The electrically protein-manipulated, microcantilever biosensor is featured with enhanced capture antibody immobilization, miniaturization, and high sensitivity. Thanks to the electric property of biological substances in a real environment, given charged proteins can be manipulated with attraction in solution under an electric field. It is evident that higher amount of capture antibody molecules immobilized onto sensing surfaces captures or detects specific molecules, indicating greater deflection and stresses as well. This however leads to significant cost in biosensors. With the merit of MEMS technique that allows highly fabrication-compatible integration into microcantilever biosensors, sparsely distributed antibody molecules in solution are attracted in focus onto a sensing solid surface under electric fields. As the sensing element of the gold-coated, V-shaped silicon nitride microcantilever also serves as an electrode, the electric fields are applied in a channel of flowing microfluidics by locally in-plane electrodes or by a top electrode arranged for three-dimensional fields. As expected, most charged proteins distributed in solution are effectively attracted onto the sensing area within the electric fields. This improves the efficiency of capture antibody immobilization and achieves an eight-fold reduction over the necessary amount of capture antibodies without applying electric fields. With such a successful manipulation of charged proteins, the novel microcantilever biosensor exhibits efficient use of capture antibodies in solution.
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    We have measured the response of gold-coated silicon nitride microcantilevers to 2-mercaptoethanol (HS–CH2–CH2–OH) vapors and found that they respond rather sensitively to 2-mercaptoethanol vapors. Such microcantilevers can be used to develop sensitive micromechanical chemical sensors for detecting sulfur-containing compounds. Microcantilevers with a gold coating on one side respond to adsorption of molecules by changes in their resonance frequency and/or bending. The bending induced from adsorption of 2-mercaptoethanol molecules on microcantilevers is at least an order of magnitude more sensitive than any corresponding resonance frequency changes resulting from mass loading. In the present studies, we used the adsorption-induced bending and placed a lower limit in the minimum detectable concentration of mercaptan vapor at <50 parts per billion (ppb).
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    We report the binding kinetics of fish-infected grouper nervous necrosis viruses (NNV) and selected antimicrobial peptides (AMPs) by nanomechanical detection. AMPs, the vital member in an innate immunity, are promising candidates in the fight against pathogens due to their broad range of antimicrobial activity and low toxicity. Grouper NNV primarily cause mass mortality of many marine cultured fish species, and two selected AMPs in this study were found to inhibit viruses by agglutinating its virions to form aggregates. The binding activity of NNVs with functionalized AMPs onto a sensing microcantilever yielded induced surface stresses, indicating high binding strength of molecular interaction. The binding affinity and kinetic rate constants of molecular recognition events calculated for NNV-AMP(TH1-5) compared to NNV-AMP(cSALF) were found to be 2.1-fold and 4.43-fold, respectively, indicating TH1-5 effectively bind with NNV more than cSALF. Moreover, a microscopic X-ray photoelectron spectroscopy technique was employed for further validation of pre- and post-NNV binding onto peptides-functionalized sensing surface. An increase in the spectrum and intensity of the P 2p and N 1s elements for the post-NNV binding was clearly shown to ensure the existence of phosphate groups and nitrogen-containing ring structures of specific NNV-TH1-5 interaction. Therefore, the microcantilever biosensing technique provides a potential and useful screening of AMPs for affinity to NNVs.
  • Article
    Nanomechanical cantilever sensors have been emerging as a key device for real-time and label-free detection of various analytes ranging from gaseous to biological molecules. The major sensing principle is based on the analyte-induced surface stress, which makes a cantilever bend. In this letter, we present a membrane-type surface stress sensor (MSS), which is based on the piezoresistive read-out integrated in the sensor chip. The MSS is not a simple "cantilever," rather it consists of an "adsorbate membrane" suspended by four piezoresistive "sensing beams," composing a full Wheatstone bridge. The whole analyte-induced isotropic surface stress on the membrane is efficiently transduced to the piezoresistive beams as an amplified uniaxial stress. Evaluation of a prototype MSS used in the present experiments demonstrates a high sensitivity which is comparable with that of optical methods and a factor of more than 20 higher than that obtained with a standard piezoresistive cantilever. The finite element analyses indicate that changing dimensions of the membrane and beams can substantially increase the sensitivity further. Given the various conveniences and advantages of the integrated piezoresistive read-out, this platform is expected to open a new era of surface stress-based sensing.
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    The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK(a), antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK(a) is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab(2) antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.
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    The alarming growth of the antibiotic-resistant superbugs methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) is driving the development of new technologies to investigate antibiotics and their modes of action. We report the label-free detection of vancomycin binding to bacterial cell wall precursor analogues (mucopeptides) on cantilever arrays, with 10 nM sensitivity and at clinically relevant concentrations in blood serum. Differential measurements have quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues. Moreover, by systematically modifying the mucopeptide density we gain new insights into the origin of surface stress. We propose that stress is a product of a local chemical binding factor and a geometrical factor describing the mechanical connectivity of regions activated by local binding in terms of a percolation process. Our findings place BioMEMS devices in a new class of percolative systems. The percolation concept will underpin the design of devices and coatings to significantly lower the drug detection limit and may also have an impact on our understanding of antibiotic drug action in bacteria.
  • Four different homogeneous non-isotopic immunoassays for the determination of total phenytoin in serum were evaluated and compared with a gaschromatographic method (GC) described by W. R. Külpmann & M. Oellerich [1981), J. Clin. Chem. Clin. Biochem. 19, 249-258) enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA), nephelometric inhibition immunoassay (NIIA) and substrate labeled fluorescent immunoassay (SLFIA). The between-days coefficients of variation in the medium therapeutic range were 4.0% (n = 29) with EMIT, 4.6% (n = 15) with FPIA, 7.8% (n = 10) with NIIA, 2.8% (n = 12) with SLFIA and 5.7% (n = 15) with GC. The recovery in spiked serum samples (phenytoin concentration: 11.9-99.1 mumol/l) was 98-101% with EMIT, 97-107% with FPIA, 102-110% with NIIA, 94-97% with SLFIA and 97-104% with GC. All of the tested immunoassays and GC yielded comparable results. The NIIA showed a somewhat lower correlation. In samples from an uraemic patient, however, great deviations from GC values were obtained with EMIT (bias: +22 to +85%) NIIA (+68 to +114%) and SLFIA (+48 to +52%). Only the results of FPIA were in good agreement with those of GC (bias: +1 to -7%). All the immunoassays showed a cross-reaction with 5-(4-hydroxyphenyl)-5-phenylhydantoin, which was most expressed with SLFIA and NIIA. The detectability of the immunoassays was adequate to allow precise measurements within the therapeutic range. After ultrafiltration of the serum, free phenytoin was measured by EMIT, FPIA and capillary gas chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
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    Discussion and development of standards for appropriate monitoring led to the following key recommendations for ordering, sampling, and analyzing antiepileptic drugs: Monitoring should usually be done on trough specimens after steady-state has been reached and always with an appropriate medical indication; non-steady-state concentrations may be indicated in selected situations. Monitoring of free phenytoin and free valproic acid is indicated in specific situations and should be done in serum. The metabolite of primidone, phenobarbital, should be measured concurrently with parent drug, but the active metabolite of carbamazepine does not need to be monitored unless the patient is exhibiting an unusual toxic response that cannot be otherwise explained. Assays used for antiepileptic drug monitoring should display a long-term CV of <10% and preferably <5%. Subtherapeutic and supratherapeutic drug concentrations should be investigated on a regular basis as part of a quality assurance process.
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    Despite the fact that all new anticonvulsants have undergone extensive pharmacokinetic scrutiny prior to their introduction to the market, the opportunity to perform good prospective studies on their concentration-effect relationship has been largely missed, in some cases deliberately because therapeutic drug monitoring (TDM) is considered unfavourable for the marketing of a new drug. However, there are reasons to believe that TDM may play a useful role in maximising the therapeutic potential of new anticonvulsants. In fact, these drugs have a narrow therapeutic index, careful individualisation of dosage to optimise response is required, and inter- and intra-individual pharmacokinetic variability may translate into differences in dosage requirements. The wide interindividual variability in the serum concentrations at which therapeutic and toxic effects of these drugs are observed does not necessarily imply that TDM cannot be useful: indeed, a marked pharmacodynamic variability has also been reported for all the currently monitored older anticonvulsants. The new anticonvulsants which, based on their properties, are particularly attractive candidates for TDM include lamotrigine, topiramate, tiagabine, zonisamide and felbamate. However, in the absence on sound information on the target concentration ranges of these drugs, the routine concentration monitoring of these drugs cannot be recommended. Despite this, serial measurements of serum drug concentrations may be useful in selected patients, especially those suspected of poor compliance and those in whom pharmacokinetic changes caused by disease or administration of concomitant medication are anticipated. Even in the presence of marked interindividual pharmacodynamic variability, it is often possible to empirically determine the concentration at which each patient exhibits the best response, and apply that information in subsequent management. Prospective studies, using preferably arandomised concentration-controlled design, are necessary to better characterise concentration-effect relationships for these agents.
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    We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A-immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.
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    Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.
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    Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.
  • Article
    A potential novel binding assay based on binding-driven micromechanical motion is described. A membrane preparation containing 5-HT(3AS) receptors was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (5-hydroxytryptamine, which is also called serotonin) or the antagonist MDL-72222, but not to other similar molecules. Control experiments show that cantilevers modified by membrane preparations that do not contain 5-HT(3AS) receptors do not respond to serotonin or MDL-72222. K(d) values obtained for serotonin and MDL-72222 are identical to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label-free, drug screening applications.
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    The aim of the present paper is to provide information concerning the setting up and interpretation of therapeutic drug monitoring (TDM) for anti-epileptic drugs. The potential value of TDM for these drugs (including carbamazepine, clobazam, clonazepam, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, pheneturide, phenobarbital, phenytoin, primidone, tiagabine, topiramate, valproic acid, vigabatrin and zonisamide) is discussed in relation to their mode of action, drug interactions and their pharmacokinetic properties. The review is based upon available literature data and on observations from our clinical practice. Up until approximately 15 years ago anti-epileptic therapeutics were restricted to a very few drugs that were developed in the first half of the 20th century. Unfortunately, many patients were refractory to these drugs and a new generation of drugs has been developed, mostly as add-on therapy. Although the efficacy of the newer drugs is no better, there is an apparent improvement in drug tolerance, combined with a diminished potential for adverse drug interactions. All new anticonvulsant drugs have undergone extensive clinical studies, but information on the relationship between plasma concentrations and effects is scarce for many of these drugs. Wide ranges in concentrations have been published for seizure control and toxicity. Few studies have been undertaken to establish the concentration-effect relationship. This review shows that TDM may be helpful for a number of these newer drugs.
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    We report an electro-mechanical biosensor for electrical detection of proteins with disease markers using self-sensing piezoresistive micro-cantilevers. Electrical detection, via surface stress changes, of antigen-antibody (Ag-Ab) specific binding was accomplished through a direct nano-mechanical response of micro-fabricated self-sensing micro-cantilevers. A piezoresistive sensor measures the film resistance variation with respect to surface stress caused by biomolecules specific binding. When specific binding occurred on a functionalized Au surface, surface stress was induced throughout the cantilever, resulting in cantilever bending and resistance change of the piezoresistive layer. The cantilever biosensors were used for the detection of prostate specific antigen (PSA) and C-reactive proteins (CRP), which are a specific marker of prostate cancer and cardiac disease. From the above experiment, it was revealed that the sensor output voltage was proportional to the injected antigen concentration (without antigen, 10 ng/ml, 100 ng/ml, 1 microg/ml). PSA and CRP antibodies were found to be very specific for their antigens, respectively. This indicated that the self-sensing micro-cantilever approach is beneficial for detecting disease markers, and our piezoresistive micro-cantilever sensor system is applicable to miniaturized biosensor systems.
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    A microfabricated cantilever with an internal piezoresistive component has been sensitized with thiol tethered ss-DNA strands and utilized for an in situ, label-free, highly specific, and rapid DNA detection assay. The generation of a differential surface stress onto the functionalized cantilever surface upon target recognition has allowed nanomechanical identification of 12-nucleotide complementary DNA probes with single base mismatch discrimination (sensitivity of 0.2 microM). Interestingly, utilization of an overhang extension distal to the surface enhanced the sensitivity to the 0.01 microM level. The cantilever was functionalized by inkjet printing technology. Replacing the capture probe with locked nucleic acid (LNA) resulted in a faster target probe capture kinetics compared to DNA-DNA hybridization. The capabilities of the piezoresistive cantilever indicate future ergonomic convenience via miniaturization alternative to the conventional laser-based detection method for portable on-site applications.
  • Article
    A promising approach for detecting biomolecules follows their binding to immobilized probe molecules on microfabricated cantilevers; binding causes surface stresses that bend the cantilever. We measured this deflection, which is on the order of tens of nanometers, by embedding a metal-oxide semiconductor field-effect transistor (MOSFET) into the base of the cantilever and recording decreases in drain current with deflections as small as 5 nanometers. The gate region of the MOSFET responds to surface stresses and thus is embedded in silicon nitride so as to avoid direct contact with the sample solution. This approach, which offers low noise, high sensitivity, and direct readout, was used to detect specific binding events with biotin and antibodies.
  • Article
    Therapeutic drug monitoring (TDM) and pharmacogenetic tests play a major role in minimising adverse drug reactions and enhancing optimal therapeutic response. The response to medication varies greatly between individuals, according to genetic constitution, age, sex, co-morbidities, environmental factors including diet and lifestyle (e.g. smoking and alcohol intake), and drug-related factors such as pharmacokinetic or pharmacodynamic drug-drug interactions. Most adverse drug reactions are type A reactions, i.e. plasma-level dependent, and represent one of the major causes of hospitalisation, in some cases leading to death. However, they may be avoidable to some extent if pharmacokinetic and pharmacogenetic factors are taken into consideration. This article provides a review of the literature and describes how to apply and interpret TDM and certain pharmacogenetic tests and is illustrated by case reports. An algorithm on the use of TDM and pharmacogenetic tests to help characterise adverse drug reactions is also presented. Although, in the scientific community, differences in drug response are increasingly recognised, there is an urgent need to translate this knowledge into clinical recommendations. Databases on drug-drug interactions and the impact of pharmacogenetic polymorphisms and adverse drug reaction information systems will be helpful to guide clinicians in individualised treatment choices.
  • Article
    To investigate pattern and extent of adverse drug reactions (ADRs) associated with AEDs and to identify safer options for treatment of epilepsy. Study was a retrospective, cross-sectional survey. Data from patients with epilepsy at the out-patient and in-patient of Neurology Department was collected in a specially designed proforma. Causality and severity of ADRs was categorized as per WHO guidelines. Among 788 patients with epilepsy, 80 (10.27%) had ADRs. ADRs with AED monotherapy were 9.18% and with polytherapy were 11.56%. ADRs with conventional and newer AED monotherapy was 10.24% and 6.84%, respectively, and were maximum with phenytoin and clobazam (14.28% and 12.5%). ADRs were mild in 4.16%, moderate in 70.83% and severe in 25% patients. Causality was probable in 65.62%, possible in 13.54% and definite in 20.83%. Patients (15/80) were hospitalized due to ADRs. Age and gender distribution showed statistically significant difference in occurrence of ADRs (p < 0.05). Chi-square test for poly versus monotherapy and conventional versus newer AEDs did not show any significant difference. Study showed maximum ADRs with AED polytherapy with no significant difference in frequency and severity of ADRs between conventional versus newer AEDs. This finding needs further investigation in larger number of patients to identify safer treatment options for epilepsy.