A computational reaction-diffusion model for the analysis of transport-limited kinetics

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigen-antibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusion-limited kinetic data.     

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.     

Engineering nanostructured porous SiO2 surfaces for bacteria detection via "direct cell capture"

An optical label-free biosensing platform for bacteria detection ( Escherichia coli K12 as a model system) based on nanostructured oxidized porous silicon (PSiO(2)) is introduced. The biosensor is designed to directly capture the target bacteria cells on its surface with no prior sample processing (such as cell lysis). The optical reflectivity spectrum of the PSiO(2) nanostructure displays Fabry-Pe?rot fringes characteristic of thin-film interference, enabling direct, real-time observation of bacteria attachment within minutes. The PSiO(2) optical nanostructure is synthesized and used as the optical transducer element. The porous surface is conjugated with specific monoclonal antibodies (immunoglobulin G's) to provide the active component of the biosensor. The immobilization of the antibodies onto the biosensor system is confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, fluorescent labeling experiments, and refractive interferometric Fourier transform spectroscopy. We show that the immobilized antibodies maintain their immunoactivity and specificity when attached to the sensor surface. Exposure of these nanostructures to the target bacteria results in "direct cell capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the biosensor. Our preliminary studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations. The current detection limit of E. coli K12 bacteria is 10(4) cells/mL within several minutes.     

Nanoscale affinity chip interface for coupling inhibition SPR immunosensor screening with Nano-LC TOF MS

The on-line nanoscale coupling of a surface plasmon resonance (SPR)-based inhibition biosensor immunoassay (iBIA) for the screening of low molecular weight molecules with nano-liquid-chromatography electrospray ionization time-of-flight mass spectrometry (nano-LC ESI TOF MS) for identification is described. The interface is based on a reusable recovery chip (RC) that contains a nanoscale biosorbent composed of a hydrogel layer modified with antibodies raised against the analyte featuring the unique possibility of performance characterization using the SPR biosensor. Various hydrogel chemistries were evaluated, and the standard Biacore CM5 chip showed the highest capture capacity in combination with affinity-purified polyclonal antibodies. The procedure has four stages: the samples are prepared (1) and screened using a screening chip (SC) in the iBIA (2). Suspected noncompliant samples as being noncompliant are reinjected over the RC, and the analyte is captured at subnanogram level (3). The captured analyte is released, and the eluate is analyzed with nano-LC ESI TOF MS via a loop-type interface (4). The coupling of the technologies proved effective for screening enrofloxacin, a model compound, in incurred chicken muscle samples followed by identity confirmation in suspected noncompliant samples. Ciprofloxacin, a known metabolite of enrofloxacin, was identified as well in incurred chicken samples. This demonstrates the potential of the technologies coupled by means of a RC for the rapid screening and identification of known as well as unknown compounds. Finally, we demonstrate the feasibility of combining the two biosensor chips (SC and RC) with a robust chip-based nano-LC chip TOF MS system, thus providing a robust alternative triple-chip system.     

Detection of chemicals by a reporter immunoassay: application to fluoride

This report describes a concept in which an immunoassay is used indirectly to quantify a nonantigenic very low molecular weight compound participating in a chemical reaction with a haptenic reporter. The detection limit of each reagent is, therefore, governed only by the affinity of the antibodies toward the reporter. Fluoride was used as a model, and silylated estradiol was used as a reporter. Upon silylation with N-O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-O-bis(dimethylterbutylsilyl) trifluoroacetamide (MTBSTFA), estradiol is no longer recognized by antibodies specific to estradiol. After reaction with hydrofluoric acid (HF) or fluoride salts (KF, CsF, NaF), its immunoreactivity is restored, and native estradiol is formed and is detected by immunoassay. The level of synthesized estradiol is dependent on the concentration of fluoride. A fluoride detection limit of 0.3 microg/L (15 nM) is obtained. Potential interference with other acids has been eliminated by choosing the silyl group (trimethylsilyl vs tert-butyldimethylsilyl) and by selecting optimal reaction conditions for the desilylation. The method has been applied to the detection of fluoride salts in natural waters (range 0.28-9.0 mg/L) and in an atmosphere artificially contaminated with HF between 8 and 160 microg/m(3) in the parts-per-billion range. This indirect immunoassay combines simplicity and high sensitivity and, therefore, can be used in field monitoring. Finally, the extension of the concept to other chemicals is discussed.     

Development of an ultrasensitive immunoassay for rapid measurement of okadaic acid and its isomers.

This report highlights the characteristics of an okadaic acid immunoassay with limits of detection in the subfemtomole range. Two different immunoassay formats were investigated and their characteristics compared in relation to linear ranges, limits of detection, and cross-reactivity with other seafood toxins present in water and/or mussel samples. The developed ELISA system can be manipulated to quantitatively measure total diarrhetic shellfish poisoning (DSP) content or for okadaic acid and dinophysistoxin-1 individual concentrations by variation of the format of the immunoassay. Real mussel samples were validated in percentage recovery test. Calibration curves were established, and aliquots of real samples were tested. Very good recoveries were attained, highlighting the validity of the ELISA system to accurately determine the DSP concentration in mussel samples.     

Ultraspecific and Highly Sensitive Nucleic Acid Detection by Integrating a DNA Catalytic Network with a Label‐Free Microcavity

Nucleic acid detection with label‐free biosensors circumvents costly fluorophore functionalization steps associated with conventional assays by utilizing transducers of impressive ultimate detection limits. Despite this technological prowess, molecular recognition at a surface limits the biosensors' sensitivity, specificity, and reusability. It is therefore imperative to integrate novel molecular approaches with existing label‐free transducers to overcome those limitations. Here, we demonstrate this concept by integrating a DNA strand displacement circuit with a micron‐scale whispering gallery mode (WGM) microsphere biosensor. The integrated biosensor exhibits at least 25‐fold improved nucleic acid sensitivity, and sets a new record for label‐free microcavity biosensors by detecting 80 pM (32 fmol) of a 22nt oligomer; this improvement results from the catalytic behavior of the circuit. Furthermore, the integrated sensor exhibits extremely high specificity; single nucleotide variants yield 40‐ to 100‐fold lower signal. Finally, the same physical sensor was demonstrated to alternatingly detect 2 different nucleic acid sequences through 5 cycles of detection, showcasing both its reusability and its versatility.     

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.     

Potentiometric biosensor employing catalytic antibodies as the molecular recognition element

Catalytic antibodies are introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the catalytic reaction of the substrate. Consequently, molecular recognition by catalytic antibodies can yield reversible immunoblosensors. In this example, a prototype potentiometric biosensor is described in which a micro-pH electrode is modified with a catalytic antibody that catalyzes the hydrolysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response is linear with the log of substrate concentration over a range of 20-500 microM with a detection limit of 5 microM under the conditions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.     

Development of xMAP Assay for Detection of Six Protein Toxins

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.     

Graphene Oxide‐Assisted and DNA‐Modulated SERS of AuCu Alloy for the Fabrication of Apurinic/Apyrimidinic Endonuclease 1 Biosensor

Fabrication of high‐performance surface‐enhanced Raman scattering (SERS) biosensors relies on the coordination of SERS substrates and sensing strategies. Herein, a SERS active AuCu alloy with a starfish‐like structure is prepared using a surfactant‐free method. By covering the anisotropic AuCu alloy with graphene oxide (GO), enhanced SERS activity is obtained owing to graphene‐enhanced Raman scattering and assembly of Raman reporters. Besides, stability of SERS is promoted based on the protection of GO to the AuCu alloy. Meanwhile, it is found that SERS activity of AuCu/GO can be regulated by DNA. The regulation is sequence and length dual‐dependent, and short polyT reveals the strongest ability of enhancing the SERS activity. Relying on this phenomenon, a SERS biosensor is designed to quantify apurinic/apyrimidinic endonuclease 1 (APE1). Because of the APE1‐induced cycling amplification, the biosensor is able to detect APE1 sensitively and selectively. In addition, APE1 in human serum is analyzed by the SERS biosensor and enzyme‐linked immunosorbent assay (ELISA). The data from the SERS method are superior to that from ELISA, indicating great potential of this biosensor in clinical applications.     

Amperometric thick-film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophosphorus hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.     

Single-layer graphene-based surface plasmon resonance sensor with dynamic evanescent field enhancement for biomarker study

ABSTRACT

In this work, we have developed a kind of single-layer graphene-based surface plasmon resonance (SLG-SPR) biosensor to detect C-reactive protein (CRP) and Prostate-specific antigen (PSA). In the experiment of testing CPR, the results obtained revealed that the changes in resonance wavelength of SLG-SPR biosensors are higher than that of the gold-film based SPR (Au-SPR) biosensors. Moreover, for the experiment of testing PSA, due to the dynamic evanescent field enhancement produced by a strong electric field coupling between the localized SPR (LSPR) of AuNPs and SPR of single-layer graphene-based film (SLG-film) that further amplify the evanescent field signal. We verified the SLG-SPR biosensors exhibited higher sensitivity than the Au-SPR biosensors and the SLG-SPR biosensor exceeded the traditional biosensor detection limit. Accordingly, the SLG-SPR biosensor based on dynamic optical enhancement can realize high sensitivity detection of low concentration biomarkers and can be applied to most of the trace biomarkers in theory.     

ssDNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes

Retinol binding protein 4 (RBP4) is a useful biomarker in the diagnosis of type 2 diabetes since its level in the serum is higher in insulin-resistant states. Accurate measurement of the serum RBP4 levels is hampered by conventional immunologic methods, such as enzyme-linked immunosorbent assay (ELISA). In this study, therefore, we have developed an aptamer-based surface plasmon resonance (SPR) biosensor that can be used to sense for RBP4 in serum samples. A single-stranded DNA (ssDNA) aptamer that showed high affinity (Kd = 0.2 +/- 0.03 microM) and specificity to RBP4 was selected. This RBP4-specific aptamer was immobilized on a gold chip and used in a label-free RBP4 detection using SPR. Analysis of RBP4 in artificial serum using SPR was compared with ELISA and Western blot analysis. Our results indicated that the RBP4-specific aptamer-based SPR biosensor gave better dose-dependent responses and was more sensitive than ELISA assays. As such, this RBP4 aptamer-based SPR biosensor can be potentially used to monitor the RBP4 levels within the serum as an indicator of type 2 diabetes.     

Mixed ceria-based metal oxides biosensor for operation in oxygen restrictive environments

The unique catalytic, electrochemical, and oxygen storage properties of ceria and mixed ceria/titania hybrid composites were used to fabricate a new type of electrochemical enzyme biosensor. These materials provided increased analytical performance and possibilities for operation in oxygen-free conditions of an oxidase enzyme biosensor using tyrosinase as a model example. The investigation of the enzymatic reaction in the presence and absence of oxygen was first carried out using cyclic voltammetry. The results were used to identify the role of each metal oxide in the immobilization matrix and fabricate a simple amperometric tyrosinase biosensor for the detection of phenol and dopamine. The biosensor was optimized and characterized with respect to response time, detection limit, linear concentration range, sensitivity, and kinetic parameters. The detection limit for phenol was in the nanomolar range, with a detection limit of 9.0 x 10(-9) M and a sensitivity of 86 mA M(-1) in the presence of oxygen and of 5.6 x 10(-9) M and a sensitivity of 65 mA M(-1) in the absence of oxygen. The optimized biosensor also showed selective determination of the neurotransmitter dopamine with a detection limit of 3.4 x 10(-8) M and a sensitivity of 14.9 mA M(-1) in the presence of oxygen and of 4.2 x 10(-8) M and 14.8 mA M(-1) in the absence of oxygen. This strategy shows promise for increasing the sensitivity of oxidase enzyme sensors and provides opportunities for operation in oxygen limited conditions. It can also be extended for the development of other enzyme biosensors.     

Glucose and lactate biosensors for scanning electrochemical microscopy imaging of single live cells

We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM) and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, and increased reproducibility for electropolymerization. We have acquired amperometric approach curves (ACs) for each type of manufactured biosensor UME, and these ACs can be used as a means of positioning the UME above a substrate at a known distance. We have used the glucose biosensor UMEs to record profiles of glucose uptake above individual fibroblasts. Likewise, we have employed the lactate biosensor UMEs for recording the lactate production above single cancer cells with the SECM. We also show that oxygen respiration profiles for single cancer cells do not mimic cell topography, but are rather more convoluted, with a higher respiration activity observed at the points where the cell touches the Petri dish. These UME biosensors, along with the application of others already described in the literature, could prove to be powerful tools for mapping metabolic analytes, such as glucose, lactate, and oxygen, in single cancer cells.     

Integrating waveguide biosensor

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.     

An immunoassay for small analytes with theoretical detection limits

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.     

Layer-by-layer assemblies of chitosan/multi-wall carbon nanotubes and glucose oxidase for amperometric glucose biosensor applications

A novel amperometric glucose biosensor based on multilayer films containing chitosan, multi-wall carbon nanotubes (MWCNTs) and glucose oxidase (GOD) was developed. MWCNTs were solubilized in chitosan (Chit-MWCNTs) used to interact with GOD. Poly (allylamine) (PAA) and polyvinylsulfuric acid potassium salt (PVS) were alternately deposited on the cleaned Pt electrode surface ((PVS/PAA)₃/Pt). The (PVS/PAA)₃/Pt electrode was alternately immersed in Chit-MWCNTs and GOD to assemble different layers of multilayer films. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Micrographs of MWCNTs were obtained by scanning electron microscope, and properties of the resulting biosensors were measured by electrochemical measurements. Among the resulting biosensors, the biosensor based on eight layers of multilayer films was best. The resulting biosensor was able to efficiently monitor glucose, with the response time within 8 s, a detection limit of 21 μM estimated at a signal-to-noise ratio of 3, a linear range of 1–10 mM, the sensitivity of 0.45 μA/mM, and well stability. The study can provide a feasible simple approach on developing a new immobilization matrix for biosensors and surface functionalization.     

Biosensor immunosurface engineering inspired by B-cell membrane-bound antibodies: modeling and analysis of multivalent antigen capture by immobilized antibodies

Immobilized antibodies are used by many biosensors and diagnostic tests as specific receptors for the presence of targeted substances in clinical, biological, or environmental samples. The antibodies used in these devices are the soluble form of the antibodies presented on the B-cell membrane: they have the same specificity, but they may differ from those presented on the B cell in orientation, flexibility, mobility, and support-membrane properties. These properties influence the formation of noncovalent bonds between the pathogen antigenic determinants (epitopes) and the amino acids of the antibodies. This paper extends the theoretical modeling foundation addressing multivalent antigen binding to cell surface receptors to account for local and far-field antibody surface density effects, immobilized antibodies, and the flexibility and range of motion of immobilized antibodies. An analysis of the derived model provides insight into the design of biosensor immunosurfaces to enhance pathogen capture capability.