Serum-based ALYGNSA immunoassay for the prostate cancer biomarker, total prostate-specific antigen (tPSA)

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced by the prostate in prostate cancer (≥4 ng/mL in the bloodstream). An immunoassay for total PSA (tPSA) was developed using the ALYGNSA method to enhance capture antibody orientation and a limit of detection of 0.63 ng/mL was reported, a limit 15-fold lower than a commercial tPSA ELISA assay. This ALYGNSA assay, however, was performed using only buffer-based proteins and blocking agents (Mackness et al., Anal Bioanal Chem 396:681–686, 2010). To improve the clinical application of this system, a serum-based tPSA ALYGNSA was developed employing human serum. This assay also resulted in a limit of detection of 0.63 ng/mL of tPSA protein. The findings reported here provide support for the clinical application of this assay for diagnosis, progression, treatment, and possible recurrence of prostate cancer.     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

Double‐Layer Nanogold and Poly(amidoamine) Dendrimer‐ Functionalized PVC Membrane Electrode for Enhanced Electrochemical Immunoassay of Total Prostate Specific Antigen

A simple and portable electrochemical immunosensor for the detection of total prostate specific antigen (t‐PSA) in human serum was developed using a double‐layer nanogold particles and dendrimer‐functionalized polyvinyl chloride (PVC) membrane as immunosensing interface. To fabricate such a multifunctional PVC electrode, an o‐phenylenediamine‐doped PVC membrane was initially constructed, then nanogold particles and poly(amidoamine) G4‐dendrimer with a sandwich‐type format were assembled onto the PVC membrane surface, and then t‐PSA antibodies (anti‐PSA) were adsorbed on the nanogold surface. The detection principle of the immunosensor is based on the change in the electric potential before and after the antigen‐antibody interaction. The experimental conditions and the factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the proposed immunosensor exhibits good electrochemical behavior in the dynamic range of 0.5–18 ng/mL relative to t‐PSA concentration with a relative low detection limit of 0.1 ng/mL (S/N=3). The precision, reproducibility, and stability of the immunosensor are acceptable. In addition, 43 serum specimens were assayed by the as‐prepared immunosensor, and consistent results were obtained in comparison with those obtained by the standard enzyme‐linked immunosorbent assay (ELISA). Compared with the conventional ELISAs, the developed immunoassay system was simple and rapid without labeling and separation steps. Importantly, the immobilization and detection methodologies could be extended for the immobilization and detection of other biomarkers.     

Monitoring of troponin release from cardiomyocytes during exposure to toxic substances using surface plasmon resonance biosensing

Troponin T (TnT) is a useful biomarker for studying drug-induced toxicity effects on cardiac cells. We describe how a surface plasmon resonance (SPR) biosensor was applied to monitor the release of TnT from active HL-1 cardiomyocytes in vitro when exposed to cardiotoxic substances. Two monoclonal human TnT antibodies were compared in the SPR immunosensor to analyse the TnT release. The detection limit of TnT was determined to be 30 ng/ml in a direct assay set-up and to be 10 ng/ml in a sandwich assay format. Exposure of the cardiomyocytes to doxorubicin, troglitazone, quinidine and cobalt chloride for periods of 6 and 24 h gave significant SPR responses, whereas substances with low toxicity showed insignificant effects (ascorbic acid, methotrexate). The SPR results were verified with a validated immunochemiluminescence method which showed a correlation of r ² = 0.790.     

Development,validation and application of a UHPLC–MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma

A UHPLC–MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C₁₈ column (100 × 2.1 mm, 2.6 μm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400 μl/min within 4.0 min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2–1,000 ng/ml with a lower limit detection of 0.5 ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.     

Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity

Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.     

Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well’s bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu–biotin binding densities of 0.080 and 0.47 pmol/mm², respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay. Figure The condensation of the binding area into a spot (right) results in a denser collection of the labeled antibodies and more favorable signal-to-background ratios compared with a regular approach using a large binding area (left)     

Aberrant sialylation of a prostate-specific antigen: Electrochemical label-free glycoprofiling in prostate cancer serum samples

Electrochemical detection method allowing to detect prostate-specific antigen (PSA), a biomarker of prostate cancer (PCa), with PSA glycoprofiling was applied in an analysis of PCa serum samples for the first time. Electrochemical impedance spectroscopy (EIS) as a label-free method with immobilized anti-PSA was applied for PSA detection and lectins to glycoprofile captured PSA on the same surface. A proper choice of blocking agent providing high selectivity of biosensor detection with the immobilized anti-PSA antibody was done. The biosensor could detect PSA down to 100 ag/mL with a linear concentration working range from 100 ag/mL up to 1 μg/mL, i.e. 10 orders of concentration magnitude and the sensitivity of (5.5 ± 0.2)%/decade. The results showed that a commercial carbo-free blocking solution was the best one, reducing non-specific binding 55-fold when compared to the immunosensor surface without any blocking agent applied, while allowing to detect PSA. The biosensor response obtained after addition of lectin (i.e. proportional to the amount of a particular glycan on PSA) divided by the biosensor response obtained after incubation with a sample (i.e. proportional to the PSA level in the sample) was applied to distinguish serum samples of PCa patients from those of healthy individuals. The results showed that Maackia amurensis agglutinin (MAA) recognizing α-2,3-terminal sialic acid can be applied to distinguish between these two sets of samples since the MAA/PSA response obtained from the analysis of the PCa samples was significantly higher (5.3-fold) compared to the MAA/PSA response obtained by the analysis of samples from healthy individuals. Thus, combined analysis of serological PSA levels together with PSA glycoprofiling of aberrant glycosylation of PSA (i.e. increase in the level of α-2,3-terminal sialic acid) has a potential to improve detection of PCa.     

A new procalcitonin optical immunosensor for POCT applications

A new immunosensor for the determination of procalcitonin was developed. A sandwich assay format was implemented on a polymethylmetacrylate optical biochip, opportunely shaped in order to obtain several flow channels and potentially suitable for point of care testing applications. The sandwich format makes use of two new rat monoclonal antibodies. The capture antibody was covalently immobilised on the surface of the plastic chip, and the detection antibody was labelled with DY647 dye. Different combinations of capture and detection antibodies were investigated, and particular attention was devoted in order to avoid the non-specific adsorption. A limit of detection of 0.088 mg L⁻¹ was achieved within the working range of 0.28–50 mg L⁻¹ in buffer samples. The assay was also implemented in human serum, and 0.2 and 0.7–25 mg L⁻¹ were the attained limit of detection and working range, respectively.     

Resonance energy transfer between ZnCdHgSe quantum dots and gold nanorods enhancing photoelectrochemical immunosensing of prostate specific antigen

Gold nanorods (AuNRs) integrated with ZnCdHgSe near-infrared quantum dots (AuNRs-ZnCdHgSe QDs) were successfully synthesized and characterized by transmission electron microscope, X-ray photoelectron spectroscopy, and X-ray diffraction. A glassy carbon electrode was decorated with the aforementioned AuNRs-ZnCdHgSe QDs nanocomposite, which provides a biocompatible interface for the subsequent immobilization of prostate specific antibody (anti-PSA). After being successively treated with glutaraldehyde vapor and bovine serum albumin solution, a photoelectrochemical immunosensing platform based on anti-PSA/AuNRs-ZnCdHgSe QDs/GCE was established. The photocurrent response of ZnCdHgSe QDs was tremendously improved by AuNRs due to the effect of resonance energy transfer which can be deduced from the dependence of the enhanced efficiency on the AuNRs with different length-to-diameter ratios and spectral absorption characteristics. A maximum photocurrent was obtained when the absorption spectrum of AuNRs matched well with the emission spectrum of ZnCdHgSe QDs. A photoelectrochemical immunosensor for prostate specific antigen (PSA) was achieved by monitoring the photocurrent variation. The photocurrent variation before and after being interacted with PSA solution exhibits a good linear relationship with the logarithm of its concentration (logc_PSA) in the range from 1.0 pg mL⁻¹ to 50.0 ng mL⁻¹. The detection limit of this photoelectrochemical immunosensor is able to reach 0.1 pg mL⁻¹ (S/N = 3). Determining PSA in clinical human serum was also demonstrated by using the developed anti-PSA(BSA)/AuNRs-ZnCdHgSe QDs/GCE electrode. The results were comparable with those obtained from an enzyme-linked immunosorbent assay method.     

Polymer–protein-enhanced fluoroimmunoassay for prostate-specific antigen

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced in prostate cancer, the total of which is comprised of two major forms, free and complexed. The common method for measuring of PSA is an Enzyme-linked immunosorbent assay (ELISA). Limits of detection using commercial PSA ELISA kits for free and total PSA were determined in our laboratory to be 1 and 10 ng/mL, respectively. A value of 0.10 was obtained for the free to total PSA ratio, a ratio of 0.25 being indicative of prostate cancer. A possible improvement in the sensitivity and detection limits of free and total PSA may be achieved by the ALYGNSA system (Clarizia et al., Anal Bioanal Chem 393:1531–1538, 2009). This fluorescent assay system employed a selective polymer biolinker system proven to enhance primary antibody orientation. Using this system, free and total PSA detection limits of 0.13 and 0.63 ng/mL, respectively, were realized. This amounted to an 8- and 15-fold improvement in detection limits for free and total PSA, respectively. A free to total PSA ratio of 0.20 was maintained in this study and may be useful for definitive diagnosis of prostate, as well as, other cancers.     

Determination of gamma-hydroxybutyric acid in dried blood spots using a simple GC-MS method with direct “on spot” derivatization

The objective of this study was the development of an accurate and sensitive method for the determination of gamma-hydroxybutyric acid (GHB) in dried whole blood samples using a GC-MS method. The complete procedure was optimized, with special attention on the sample pretreatment, and validated. Therefore, dried blood spots of only 50 μl were prepared and, after the addition of internal standard GHB-d6, directly derivatized using 100 μl of a freshly prepared mixture of trifluoroacetic acid anhydride and heptafluorobutanol (2:1). The derivatized extract was injected into a gas chromatograph coupled to a mass spectrometer (GC-MS), operating in the electron impact mode, with a total run time of 12.3 min. Method validation included the evaluation of linearity, precision, accuracy, sensitivity, selectivity, and stability. A weighting factor of 1/x ² was chosen and acceptable intra-batch precision, inter-batch precision, and accuracy were seen. The linear calibration curve ranged from 2 to 100 μg/ml, with a limit of detection of 1 μg/ml. Our procedure, utilizing the novel approach of direct “on spot” derivatization followed by analysis with GC-MS, proved to be reliable, fast, and applicable in routine toxicology.     

A new strategy for label-free electrochemical immunoassay based on “gate-effect” of β-cyclodextrin modified electrode

A novel label-free electrochemical immunoassay was developed for prostate-specific antigen (PSA) detection via using β-cyclodextrin (β-CD) assembled layer created gates for the electron transfer of probe. To construct the sensor, a gold electrode was self-assembled with monoclonal anti-PSA antibody labeled 6-mercapto-β-cyclodextrin. Interspaces among β-CD molecules in the layer were automatically formed on gold electrode, which act as the channel of the electron transfer of [Fe(CN)₆]^3−/4− probe. When PSA bind with anti-PSA, it can block these channels on the electrode surface due to their steric hindrance effect, resulting in the decrease in redox current of the probe. Through such a gate-controlled effect, ultra trace amount of PSA may make the currents change greatly after the immunoreaction, which enhanced the signal-to-noise ratio to achieve the amplification effect. By evaluating the logarithm of PSA concentrations, the immunosensor had a good linear response to the current changes with a detection limit of 0.3 pg/mL (S/N = 3) when PSA concentration ranged from 1.0 pg/mL to 1.0 ng/mL. The label-free immunosensor exhibited satisfactory performances in sensitivity, repeatability as well as specificity.     

A highly sensitive capillary electrophoresis immunoassay strategy based on dual‐labeled gold nanoparticles enhancing chemiluminescence for the detection of prostate‐specific antigen

An enzyme and antibody dual labeled gold nanoparticles enhancing chemiluminescence strategy was developed for highly sensitive CE immunoassay (IA) of prostate‐specific antigen (PSA). In this work, gold nanoparticles were labeled with horseradish peroxidase and antiprostate specific antigen‐antibody, and used as the marker (Ab^*). After PSA (antigen, Ag) was added into the system, a noncompetitive immune reaction was happen between Ab^* and Ag to form an immune complex (Ag–Ab^*). Subsequently, the obtained Ag–Ab^* and unreacted Ab^* were separated by CE, and the chemiluminescence intensity of Ag‐Ab^* was used to estimate PSA concentration. The calibration curve showed a good linearity in the range of 0.25–10 ng/mL. Based on a S/N of 3, the detection limit for PAS was estimated to be 0.092 ng/mL. Proposed CE method was applied for PSA quantification in human serum samples from healthy volunteers and patients with prostate cancer. The obtained results demonstrated that the proposed CE method may serve as an alternative tool for clinical analysis of PSA.     

Novel fluoroimmunoassay for ovarian cancer biomarker CA-125

Cancer antigen 125 (CA-125) is a glycoprotein biomarker that denotes the presence of ovarian and reproductive cancers in women, with serum concentrations of CA-125 greater than 35 U/ml considered indicative of potential malignancies. A fluorescent immunoassay recently developed in our laboratory employing the ALYGNSA antibody-orientation system has been used to measure CA-125 levels. This system displayed significantly increased sensitivity with a detection limit of 1.5 U/ml compared to that of a commercial CA-125 enzyme-linked immunosorbent assay (15 U/ml) This tenfold lower level of detection of the ALYGNSA CA-125 assay should permit better identification and monitoring of ovarian cancer.     

A novel strategy for ultra-sensitive electrochemical immunoassay of biomarkers by coupling multifunctional iridium oxide (IrO<Subscript>x</Subscript>) nanospheres with catalytic recycling of self-produced reactants

In this study, a novel and enzyme-free electrochemical immunoassay was developed for the detection of alpha-fetoprotein (AFP; as a model biomarker) by using a novel redox nanocatalyst, patterned gold-iridium oxide nanoparticle assemblies (Au-IrO_x), on a Prussian blue (PB) functionalized glassy carbon electrode. The as-prepared Au-IrO_x nanospheres were not only used for the labeling of secondary anti-AFP antibody but also executed for water oxidation. With a sandwich immunoassay format, the electrochemical measurement was carried out in 0.1 M KNO₃-HCl, pH 3.2, containing 1.0 mM p-aminophenol (AP). Initially, the added AP was oxidized to p-quinone imine (QI) with the help of the immobilized PB on the electrode, and then the generated QI was reduced back to AP by hydrogenation reaction from water oxidation. The self-produced QI reactants were catalytically recycled and thus amplified the signal of electrochemical response. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.005 to 200 ng/mL with a low detection limit of 0.5 pg/mL AFP (at 3s_B). Intra- and inter-assay coefficients of variation were below 10%. In addition, the methodology was validated with real serum samples, receiving a good agreement with the results obtained from commercially available electrochemiluminescence automated analyzer.     

Simultaneous determination of twelve biogenic amines in serum by high performance liquid chromatography

In this study, we established a method for simultaneously determining twelve biogenic amines in serum by using reversed phase high performance liquid chromatography (RP-HPLC). The biogenic amines were first extracted from human serum by perchloric acid solution and derivatized by dansyl chloride. An ODS column was selected as separation column at 40 °C. The mobile phase solutions were consisted of A, 0.1 mol/L ammonium acetate and B, acetonitrile. A gradient elution was carried out with a flow rate at 1.0 ml/ml. The results show that the detection limit for twelve biogenic amines ranged between 0.0621 and 0.628 μg/L. All the correlation coefficients were above 0.999. The linearity was over the range from 0.001 to 20 mg/L depending on individual biogenic amine. The intra-day and inter-day coefficients of variations were from 0.53% to 7.50%,and from 1.10% to 7.25% respectively. The average analytical recovery in serum was from 92.02% to 107.65%. Moreover, the serum concentrations of tryptamine, tyramine and histamine in healthy females were found lower than that in healthy males significantly. The method is sensitive, convenient, and reliable, and suitable for simultaneous analysis of multiple biogenic amines in the clinical diagnosis and drug discovery.     

Indirect Inhibitive Immunoassay for Estradiol Using Surface Plasmon Resonance Coupled to Online In-Tube SPME

Abstract

A new immunoassay method using surface plasmon resonance (SPR) coupled with online, in-tube, solid-phase microextraction (SPME) was developed and applied to detect estradiol in human serum and seawater using an indirect inhibitive immunoassay format. The binding of antibody was inhibited by estradiol in a dose-dependent manner, and the calibration curve was generated by linear fit over the range of 0.3125–20.0 ng/ml, with a correlation coefficient of R² = 0.99996. The detection limit (S/N = 3) was 0.17 ng/ml. This study demonstrates, for the first time, the feasibility of in-tube SPME-SPR to determine estradiol in human serum and seawater.