Polymer-functionalized silica nanosphere labels for ultrasensitive detection of tumor necrosis factor-alpha

A signal amplification strategy for sensitive detection of tumor necrosis factor-alpha (TNF-α) using quantum dots (QDs)-polymer-functionalized silica nanosphere as the label was proposed. In this approach, silica nanospheres with good monodispersity and uniform structure were employed as carriers for surface-initiated atom transfer radical polymerization of glycidyl methacrylate, which is readily available functional monomer that possessing easily transformable epoxy groups for subsequent CdTe QDs binding through ring-open reaction. Then, human anti rabbit TNF-α antibody (anti-TNF-α, Ab2, served as a model protein) was bonded to CdTe QDs-modified silica nanospheres coated with polymer to obtain QDs-polymer-functionalized silica nanosphere labels (Si/PGMA/QD/Ab2). The Si/PGMA/QD/Ab2 labels were attached onto a gold electrode surface through a subsequent "sandwich" immunoreaction. This reaction was confirmed by scanning electron microscopy (SEM) and fluorescence microscopic images. Enhanced sensitivity could be achieved by an increase of CdTe QD loading per immunoassay event, because of a large number of surface functional epoxy groups offered by the PGMA. As a result, the electrochemiluminescence (ECL) and square-wave voltammetry (SWV) measurements showed 10.0- and 5.5-fold increases in detection signals, respectively, in comparison with the unamplified method. The detection limits of 7.0 pg mL(-1) and 3.0 pg mL(-1) for TNF-α antibodies by ECL and SWV measurements, respectively, were achieved. The proposed strategy successfully demonstrated a simple, reproducible, specific, and potent method that can be expanded to detect other proteins and DNA.     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Amplified electrochemical detection of a cancer biomarker by enhanced precipitation using horseradish peroxidase attached on carbon nanotubes

An electrochemical nanoimmunosensor based on multiwall carbon nanotubes (MWCNTs)/gold nanoparticles (AuNPs) was developed for the amplified detection of prostate specific antigen (PSA). The amplified detection was achieved by the enhanced precipitation of 4-chloro-1-naphthol (CN) using a higher number of horseradish peroxidase (HRP) molecules attached on MWCNTs. The PSA nanoimmunosensor was fabricated by immobilizing a monoclonal anti-PSA antibody (anti-PSA) on the AuNP-attached thiolated MWCNT on a gold electrode. The sensor surface was characterized using scanning electron microscope, transmission electron microscope, quartz crystal microbalance, and electrochemical techniques. Cyclic and square wave voltammetric techniques were used to monitor the enhanced precipitation of CN that accumulated on the electrode surface and subsequent decrement in the electrode surface area by monitoring the reduction process of the Fe(CN)(6)(3-)/Fe(CN)(6)(4-) redox couple. Under the optimized experimental condition, the linear range and the detection limit of PSA immunosensor were determined to be 1.0 pg/mL to 10.0 ng/mL and 0.40 ± 0.03 pg/mL, respectively. The validity of the proposed method was compared with an enzyme-linked immunosorbent assay method in various PSA spiked human serum samples.     

Carbon nanotube microwell array for sensitive electrochemiluminescent detection of cancer biomarker proteins

This paper describes fabrication of a novel electrochemiluminescence (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests residing in the bottoms of 10-μL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)(3)](2+) and secondary antibodies (RuBPY-silica-Ab(2)) are employed in this system for highly sensitive two-analyte detection. Antibodies to prostate specific antigen (PSA) and interleukin-6 (IL-6) were attached to the same RuBPY-silica-Ab(2) particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 in. × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab(2) is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which generates excited state [Ru(bpy)(3)](2+) in the 100-nm particles, and is measured with a charge-coupled device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. The detection limit (DL) for PSA was 1 pg mL(-1) and for IL-6 was 0.25 pg mL(-1) (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to the detection of two or more proteins.     

Electrochemical immunoassay of carcinoembryonic antigen based on a lead sulfide nanoparticle label

We describe a lead sulfide nanoparticle (PbS NP)-based electrochemical immunoassay to detect a tumor biomarker, carcinoembryonic antigen (CEA). Cubic PbS NPs were prepared and functionalized with thioglycolic acid (TGA), which stabilized the formed NPs and offered carboxyl groups to conjugate with CEA antibodies. PbS NP conjugated with monoclonal CEA antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary CEA antibody (immobilized on the carboxyl-modified magnetic beads), CEA and the PbS-labeled secondary antibody (PbS-anti-CEA), PbS labels were captured to the magnetic-bead (MB) surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured PbS was used to quantify the concentration of CEA after an acid-dissolution step. The MBs and the magnetic separation platform were used to integrate a facile antibody immobilization with immunoreactions and the isolation of immunocomplexes from reaction solutions in the immunoassay. The voltammetric response is highly linear over the range of 1-50?ng?ml(-1) CEA, and the limit of detection is estimated to be 0.5?ng?ml(-1). The performance of this nanoparticle-based electrochemical immunoassay was successfully evaluated with human serum spiked with CEA, indicating that this convenient and sensitive technique offers great promise for rapid, simple and cost-effective analysis of tumor biomarkers in biological fluids.     

Electrochemical sensor for organophosphate pesticides and nerve agents using zirconia nanoparticles as selective sorbents

An electrochemical sensor for detection of organophosphate (OP) pesticides and nerve agents using zirconia (ZrO2) nanoparticles as selective sorbents is presented. Zirconia nanoparticles were electrodynamically deposited onto the polycrystalline gold electrode by cyclic voltammetry. Because of the strong affinity of zirconia for the phosphoric group, nitroaromatic OPs strongly bind to the ZrO2 nanoparticle surface. The electrochemical characterization and anodic stripping voltammetric performance of bound OPs were evaluated using cyclic voltammetric and square-wave voltammetric (SWV) analysis. SWV was used to monitor the amount of bound OPs and provide simple, fast, and facile quantitative methods for nitroaromatic OP compounds. The sensor surface can be regenerated by successively running SWV scanning. Operational parameters, including the amount of nanoparticles, adsorption time, and pH of the reaction medium have been optimized. The stripping voltammetric response is highly linear over the 5-100 ng/mL (ppb) methyl parathion range examined (2-min adsorption), with a detection limit of 3 ng/mL and good precision (RSD = 5.3%, n = 10). The detection limit was improved to 1 ng/mL by using 10-min adsorption time. The promising stripping voltammetric performances open new opportunities for fast, simple, and sensitive analysis of OPs in environmental and biological samples. These findings can lead to a widespread use of electrochemical sensors to detect OP contaminates.     

Synthesis of silver nanoparticle-hollow titanium phosphate sphere hybrid as a label for ultrasensitive electrochemical detection of human interleukin-6

A silver nanoparticle-hollow titanium phosphate sphere (AgNP-TiP) hybrid is successfully synthesized and used as a label for electrochemical detection of human interleukin-6 (IL-6). Hollow TiP spheres with a diameter of 430 nm and an average thickness of 40 nm are synthesized by a template approach. The AgNPs are incorporated in situ into the TiP shell via an exchange process. The as-prepared AgNP-TiP hybrid shows outstanding biocompatibility, good dispersity and solubility in water, and high silver loading properties (289.2 mg of silver in 1.0 g of TiP). These advantages make the AgNP-TiP hybrid an effective candidate as an amplification label in immunoassay systems. Herein, the as-prepared AgNP-TiP hybrid is attached to a signal antibody (Ab(2) ) to produce Ab(2) -AgNP-TiP labels in the fabrication of an electrochemical immunosensor. The nanoparticle-based amplification labels, upon coupling with a magnetic sensing array, give rise to an extremely sensitive response to IL-6 in a linear range of 0.0005-10 ng mL(-1) with a detection limit of 0.1 pg mL(-1) . The proposed sensor exhibits high specificity, good reproducibility, and long-term stability, and may be a promising technique for protein and DNA detection.     

A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.     

Magnetic electrochemical immunoassays with quantum dot labels for detection of phosphorylated acetylcholinesterase in plasma

A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP-AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP-AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3-300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides.     

In situ amplified electrochemical immunoassay for carcinoembryonic antigen using horseradish peroxidase-encapsulated nanogold hollow microspheres as labels

Methods based on sandwich-type electrochemical enzyme immunoassay protocol have been extensively developed for the detection of biomolecules, but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. In this study, we initially synthesized specially horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS), and then the prepared HRP-GHS was conjugated to the secondary carcinoembryonic antibody (HRP-GHS- anti-CEA). Carcinoembryonic antigen (CEA), as a model protein, was monitored by using the electrochemical sandwich-type enzyme immunoassay format. Under optimized conditions, the linear range of the immunoassay by using single HRP-labeled anti-CEA (HRP- anti-CEA) as secondary antibodies is 2.5-120 ng/mL with a detection limit of 1.5 ng/mL CEA, while the assay sensitivity by using HRP-GHS- anti-CEA as secondary antibodies is further increased from 0.01 to 200 ng/mL with a lower detection limit of 1.5 pg/mL CEA. The intra- and interassay reproducibility is acceptable. The CEA concentrations of the clinical serum specimens assayed by the developed immunoassay show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Determination of cardiac troponin I for the auxiliary diagnosis of acute myocardial infarction by anodic stripping voltammetry at a carbon paste electrode

An electrochemical immunoassay for cardiac troponin I (cTnI) combining the concepts of the dual monoclonal antibody "sandwich" principle, the silver enhancement on the nano-gold particle, and the anodic stripping voltammetry is described. Four main steps were carried out to obtain the analytical signal, i.e., electrode preparation, immunoreaction, silver enhancement, and anodic stripping voltammetric detection. A linear relationship between the anodic stripping peak current and concentration of cTnl from 1 to 20 ng/ml and a limit of detection of 0.8 ng/ml were obtained. The established method was tested by determining cTnI in acute myocardial infarction (AMI) samples using enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.     

Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using distinguishable signal tags

A novel flow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in biological fluids was designed using biofunctionalized magnetic graphene nanosheets (MGO) as immunosensing probes and multifunctional nanogold hollow microspheres (GHS) as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA (Ab(1)) and anti-AFP (Ab(2)) antibodies on the Fe(3)O(4) nanoparticle-coated graphene nanosheets (MGO-Ab(1,2)). The reverse-micelle method was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide (HRP)-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were utilized as labels of the corresponding GHS-Ab(1) and GHS-Ab(2). A sandwich-type immunoassay format was employed for the online detection of CEA and AFP by coupling a flow-through detection cell with an external magnet. The assay was based on the catalytic reduction of H(2)O(2) at the various peak potentials in the presence of the corresponding mediators. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01-200 ng mL(-1) for AFP and 0.01-80 ng mL(-1) for CEA. The detection limits (LODs) for both analytes at 1.0 pg mL(-1) (at 3s(B)) were very low. No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intraassay and interassay coefficients of variation were <10%. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the flow-through multiplexed electrochemical immunoassay and an electrochemiluminescence method as a reference.     

Electrochemical enzyme immunoassays on microchip platforms.

A microfluidic device for conducting electrochemical enzyme immunoassays is described. The new "lab-on-a-chip" protocol integrates precolumn reactions of alkaline phosphatase-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibody-antigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the 4-aminophenyl phosphate substrate and a downstream amperometric detection of the liberated 4-aminophenol product Factors influencing the reaction, separation, and detection processes were optimized, and the analytical performance was characterized. An applied field strength of 256 V/cm results in free antibody and antibody-antigen complex migration times of 125 and 340 s, respectively. A remarkably low detection limit of 2.5 x 10(-16) g/mL (1.7 x 10(-18) M) is obtained for the mouse IgG model analyte. Such combination of a complete integrated immunoassay, an attractive analytical performance, and the distinct miniaturization/portability advantages of electrochemical microsystems offers considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics or on-site environmental testing.     

Label-free electrochemical immunoassay for the detection of human chorionic gonadotropin hormone

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.     

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ～1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.     

Versatile apoferritin nanoparticle labels for assay of protein

A versatile bioassay label based on marker-loaded apoferritin nanoparticles (MLANs) has been developed for sensitive protein detection. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin provides a facile route to prepare nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. A fluorescence marker (fluorescein anion) and a redox marker [hexacyanoferrate(III)] were used as model markers to load into the cavity of apoferritin nanoparticles for microscopic fluorescence immunoassay and electrochemical immunoassay, respectively. Detection limits of 0.06 (0.39 pM) and 0.08 ng mL(-1) (0.52 pM) IgG were obtained with fluorescein MLAN and hexacyanoferrate MLANs, respectively. The new nanoparticle labels hold great promise for multiplex protein detection (in connection with nanoparticles loaded with different markers) and for enhancing the sensitivity of other bioassays.     

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.     

Rapid diagnostic barcode system for codetection of multiple protein markers

An ultrasensitive immunodiagnostic readout method based on an electrochemical analysis is presented. Different inorganic quantum dot (QD) nanocrystals (ZnS, CdS, and PbS) are tagged to antibodies for the on-site voltammetric stripping measurements of multiple antigen targets. The multiprotein electrical sensing capability is coupled to the amplification feature of anodic stripping voltammetric transduction and with an efficient magnetic removal (to minimize nonspecific adsorption and cross-reactivity effects). Sandwich-immunoassay formats were performed using model proteins (/spl beta//sub 2/-microglobulin, myoglobin, and human serum albumin). These encoding QD tracers with distinct redox potential yield highly sensitive and selective stripping peaks at -1.11 V (Zn), -0.67 V (Cd), and -0.52 V (Pb) at the mercury-film screen printed carbon electrode (versus Ag/AgCl reference). The position and size of these peaks reflect the identity and risk level of the corresponding antigen marker. The favorable signal-to-noise characteristics of the response for the initial 25-ng/mL mixture indicate a detection limit of ca. 10 ng/mL far below the early warning range and allow a reliable determination of very low protein concentrations. Such analog peaks of the QDs were converted to simple and rapid barcode signals. The digital readout system can code 215 electrically tuned barcodes to mark different protein analytes and to be useful for a wireless communication system.     

Sensitive amperometric immunosensing using polypyrrolepropylic acid films for biomolecule immobilization

An electrochemical immunosensor was constructed using an electropolymerized pyrrolepropylic acid (PPA) film with high porosity and hydrophilicity. A high density of carboxyl groups of PPA was used to covalently attach protein probes, leading to significantly improved detection sensitivity compared with conventional entrapment methods. As a model, anti-mouse IgG was covalently immobilized or entrapped in the PPA film and used in a sandwich-type alkaline phosphatase-catalyzing amperometric immunoassay with p-aminophenyl phosphate as the substrate. With covalent binding, the detection limit for IgG in PBS buffer, pH 7.4, was 100 pg/mL with a dynamic range of 5 orders of magnitude. The covalent bonding mode in the carbonate-bicarbonate buffer, pH 9.6, further brought down the detection limit to 20 pg/mL with remarkable selectivity.     

Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab(2)). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab(1)) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab(2)) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL(-1) to 8 ng mL(-1). The detection limit of PSA was 8 pg mL(-1) (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.