Prostate-specific Antigen Antibody Research Articles

Research Update: Nanoscale surface potential analysis of MoS2 field-effect transistors for biomolecular detection using Kelvin probe force microscopy

We used high-resolution Kelvin probe force microscopy (KPFM) to investigate the immobilization of a prostate specific antigen (PSA) antibody by measuring the surface potential (SP) on a MoS2 surface over an extensive concentration range (1 pg/ml–100 μg/ml). After PSA antibody immobilization, we demonstrated that the SP on the MoS2 surface characterized by KPFM strongly correlated to the electrical signal of a MoS2 bioFET. This demonstration can not only be used to optimize the immobilization conditions for captured molecules, but can also be applied as a diagnostic tool to complement the electrical detection of a MoS2 FET biosensor.

Nano-fabricated memristive biosensors for biomedical applications with liquid and dried samples.

Nanowire based platforms are widely reported for sensing applications and for their potential in the bio-detection field. In the present work, memristive nanowire devices are implemented for label-free sensing in liquid samples as well as in dried samples via leveraging the modification of the hysteresis in the devices electrical response as a consequence of the surface modification. First, pH sensing in liquid conditions is demonstrated. In addition, the memristive devices are bio-functionalized using antibodies for Prostate Specific Antigen (PSA) as case of study. In this way, the nanowires are converted to memristive biosensors paving the way for future molecular diagnostics applications in general, and for detection of prostate cancer disease in particular.

Construction of novel electrochemical immunosensor for detection of prostate specific antigen using ferrocene-PAMAM dendrimers

In this study, an immunosensor was designed to utilize for the detection of prostate specific antigen (PSA) based on three different generations (G1, G2 and G3) of ferrocene (Fc) cored polyamidiamine dendrimers (Fc-PAMAM) gold (Au) electrode. The self-assembled monolayer principle (SAM) was used to fabricate the sensitive, selective and disposable immunosensor electrodes. In electrode fabrication cysteamine (Cys) was the first agent covalently linked on the Au electrode surface. Immobilized redox center (ferrocene) cored PAMAM dendrimers served as a layer for the further binding of biological components. The monoclonal antibody of PSA (anti-PSA) was covalently immobilized on dendrimers which were attached onto the modified Au surface (Au/Cys/Fc-PAMAMs/anti-PSA). PSA levels were quantitatively analyzed by using electrochemical differential pulse voltammetry (DPV) whose lowest detection limit was calculated as 0.001ngmL−1. The Au/Cys/FcPAMAM/anti-PSA immunosensor showed excellent performance for PSA at the pulse amplitude; 50mV and the scan rate; 10mV/s in a wide linear concentration range of 0.01ng–100ngmL−1. Analytical performance and specificity assays were carried out using human serum and different proteins.

On-Chip Mass Spectrometry-Based Immunoassay as a Tool for the Detection of Molecular Species from Prostate-Specific Antigen in Female Serum

ABSTRACTIn females, low levels of prostate-specific antigen have been detected in hormonally dependent tissues as well as in serum and body fluids using immunometric assays. Enzymatically inactive free prostate-specific antigen was found to be the predominant form in sera of women with breast cancer, in contrast to sera from healthy women, but its molecular nature is not understood. In comparison to conventional immunoassays, mass spectrometry-based immunoassays are advantageous in terms of providing analytical data on both concentration and structural information regarding the analyte. This study was aimed at exploiting on-chip mass spectrometry-based immunoassay for profiling the molecular species of prostate-specific antigen in sera of women with breast cancer and healthy females to resolve presumed cancer-associated free prostate-specific antigen heterogeneity. Serum was analyzed using an immobilized anti-free prostate-specific antigen antibody array. Immunoreactive species were detected using surface-enhanced laser desorption–ionization time-of-flight mass spectrometry. The results obtained indicated similarities in profiles of the examined female sera regarding glycosylated full-length molecular species at 28 kDa, while noticeable differences were observed between the groups regarding immunoreactive species, reflecting modification of free prostate-specific antigen by proteolytic cleavage. The free prostate-specific antigen profile in breast cancer was specific for species at 12, 16, 21–23 kDa, and at 29 kDa, which might differ in glycosylation status. The results on low-abundance free prostate-specific antigen in female sera as yet unexploited biomarker direct further efforts to innovative use of conventional proteomic methods in diagnostic bioanalytical challenges.

Joint enhancement strategy applied in ECL biosensor based on closed bipolar electrodes for the detection of PSA

A highly sensitive electrogenerated chemiluminescence (ECL) biosensor was developed on the basis of a closed bipolar electrode (BPE) apparatus for the analysis of prostate specific antigen (PSA). Bipolar modifications bring up two different stages of enhancement on the same electrode. Anodic enhancement was conducted by modifying gold nanoparticles (Au NPs) to catalyze the anodic ECL reaction between luminol and hydrogen peroxide. Cathodic introduction of thionine tagged PSA antibody led to a further pertinently enhancement synchronized with the PSA amount variation, because the existence of thionine greatly increased the rate of electron gains on cathode, leading to the corresponding acceleration of anodic ECL reaction. The more thionine modified target molecules were introduced, the faster luminol was oxidized, the higher faraday current approached, and sensitive quantification was realized in correlation with the responsive ECL intensity differences. The quantification resulted in a good determination range between 0.1pg/mL and 0.1µg/mL. This strategy mainly took advantage of the special structure of closed BPE to realize a simultaneous amplification on both ends of BPE. Moreover, the platform had a potential of providing a multi-functional strategy for the realization of other bio-detections by simply substituting the PSA sandwich structure with other bio-structures.

Label-free detection of prostate specific antigen (PSA) using a bridge-shaped PZT resonator

This paper reports the results of the development and testing of a bridge-shaped resonator to detect different concentrations of prostate specific antigen (PSA) based on shifts in resonant frequency. The bridge-shaped PZT resonator was fabricated by Micro Electro Mechanical Systems techniques, including photolithography, wet and dry etching, and film deposition. The prototype consists of multiple layers comprised of Ta/Pt/PZT/Pt followed by a supporting layer of SiNx. The resonant frequency of the bridge-shaped PZT resonator was measured in the air by a heterodyne laser doppler vibrometer, and the frequencies were 406 ± 3, 177 ± 2, and 121 ± 1 kHz for the dimensions of 50 μm × 250 μm, 100 μm × 500 μm, and 150 μm × 750 μm (width × length), respectively. Bridge-shaped PZT resonators of different geometries have been used to detect PSA over a wide range of concentrations from 10 pg/ml to 100 ng/ml, which includes the clinically relevant diagnostic range. Using the specific binding between PSA and PSA antibody, different concentrations of PSA can be detected through the change of resonant frequency caused by the mass of the bound PSA. The measured resonant frequency and the calculated mass of bound PSA were used to calculate mass sensitivity (S = Δf/Δm). Mass sensitivities of bridges with dimensions of 50 μm × 250 μm, 100 μm × 500, and 150 μm × 750 μm were 44.14, 18.90 and 8.31 Hz/pg, respectively. This shows that mass sensitivity increases as the dimensions of the bridge get smaller. The feasibility of detecting 10 pg/ml of PSA was confirmed, which is a much smaller amount than 4 ng/ml, which is the conventional cut-off point between a patient and a non-patient in clinical diagnostics.

Electrochemiluminescent immunosensing of prostate-specific antigen based on silver nanoparticles-doped Pb (II) metal-organic framework

In this work, silver nanoparticles-doped Pb (II) metal-organic framework (Ag-MOF) was prepared and exploited as a luminescence probe for the development of label-free electrochemiluminescence (ECL) immunosensing scheme for prostate-specific antigen (PSA). The β-cyclodextrin based MOF, Pb-β-cyclodextrin (Pb(II)-β-CD) shows excellent ECL behavior and unexpected reducing capacity towards silver ions. Silver nanoparticles could massively form on the surface of Pb(II)-β-CD (Ag@Pb(II)-β-CD) without use any additional reducing agent, while the ECL behavior of Pb(II)-β-CD still was well retained. The Ag@Pb(II)-β-CD was used as a substrate material to modify glass carbon electrodes and formed a sensing platform for the fabricating ECL immunosensor. The presence of silver nanoparticles enables the facile immobilization of capturing antibody of PSA. The specific binding of PSA onto the electrode surface induces the decrease of ECL signals. A linear range of 0.001–50ngmL−1 with a detection limit of 0.34pgmL−1 (S/N=3) was obtained after the optimization of experimental conditions. This simply fabricated immunosensor exhibits good stability, accuracy and acceptable reproducibility, which suggesting its potential applications in clinical diagnostics.

Abstract 4344: Olfactomedin 4 plays a tumor-suppressor role and is a novel candidate biomarker in the prostate cancer progression and independent of PSA

Abstract Prostate cancer is the most commonly diagnosed solid tumor and the second leading cause of cancer-related death in the American male population. The olfactomedin 4 gene (OLFM4) plays critical tumor suppressor roles in human and murine prostate cancer. We have previously reported that loss or reduce of OLFM4 expression is associated with the progression of human prostate cancer. We further found that loss of Olfm4 leads to prostate neoplastic progression in the Olfm4-knockout mouse model. To study the clinical impacts of OLFM4 gene in the prostate cancer, we have performed bioinformatics analysis from the published datasets, GSE35988, GSE21032 and TCGA as well as immunohistochemical analysis with prostate cancer tissue arrays by using OLFM4 antibody. The copy number variations (CNV) of OLFM4 gene were significantly associated with Gleason scores of prostate cancer. The pattern of OLFM4 CNV is similar with other tumor suppressor genes, RB1, PTEN and P53. We have observed consist reduce or loss of mRNA expression in the castrate resistant and metastatic prostate cancer patient samples. To study the relationship between OLFM4 expression and prostate cancer recurrence, we performed Kaplan-Meier Plots and Log-rank statistic analysis for GSE21032 and TCGA data sets. Overall, there was no significant relationship between OLFM4 expression and prostate cancer recurrence. However, when analysis was restricted to the lower quartile of OLFM4 expression, we obtained a highly significant inverse relationship (p = 0.02) between OLFM4 expression and recurrence. We also observed that alteration of CNV and expression of OLFM4 gene has no relationship with the level of (patient) serum prostate specific antigen (PSA). We performed immunohistochemical analysis with prostate cancer tissue arrays by using OLFM4, PSA and E-cadherin antibodies. Lower or loss of expression of OLFM4 protein is associated with advanced Gleason scores of prostate cancer specimens. The pattern of OLFM4 protein expression in prostate cancer patient samples is similar to that of E-cadherin, but not PSA. Taken together, these results suggest that OLFM4 plays a tumor-suppressor role, and therefore is a likely therapeutic target for prostate cancer interdiction. Citation Format: Hongzhen Li, Ye Chen, Wenli Liu, Jianqiong Zhu, Chin Kay, Xujing Wang, Griffin P. Rodgers. Olfactomedin 4 plays a tumor-suppressor role and is a novel candidate biomarker in the prostate cancer progression and independent of PSA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2015-4344

Ultrasensitive electrochemical immunosensor for PSA biomarker detection in prostate cancer cells using gold nanoparticles/PAMAM dendrimer loaded with enzyme linked aptamer as integrated triple signal amplification strategy.

In the present study, a triple signal amplification strategy was developed for ultrasensitive immunosensing of prostate-specific antigen (PSA) tumor marker. The proposed system was achieved by modification of glassy carbon electrode with graphene oxide/chitosan film and covalently attached of monoclonal PSA antibody and thionine as redox probe onto the modified electrode surface. Then, immunosensing was completed using sandwich-type immunoreaction of the PSA-antigen between anti-PSA immobilized on the graphene/chitosan interface and PSA-aptamer. For improve the sensitivity, polyamidoamine dendrimer-encapsulated gold nanoparticles (AuNPs-PAMAM) was used for covalent attachment of PSA-aptamer and HRP linked aptamer (Au-PAMA/aptamer-HRP) as electrochemical label in the sandwich format and electrocatalytic reduction of H2O2 in the presence of enzymatically oxidized thionine was measured. Under optimized condition, the obtained detection limit and linear concentration range were 10 fg ml(-1)(S/N=3) and 0.1 pg ml(-1) to 90 ngml(-1) respectively, using differential pulse voltammetry as measuring technique. In addition, electrochemical impedance spectroscopy (EIS) was used as simple, rapid, low cost label free analytical technique for PSA measurement with detection limit of 5 pg ml(-1) at concentration range up to 35 ng ml(-1). Finally, the immunosensor is used to PSA detection in human serum and prostate tissue samples and the obtained result is well agreed with the values obtained by the standard ELISA method. The obtained results indicate the proposed immunosensor can be used for monitor the differences in PSA concentration in cancer tissue samples which holds great promise in clinical screening of cancer biomarkers.

A novel electrochemiluminescence resonance energy transfer system for ultrasensitive detection of prostate-specific antigen

A novel electrochemiluminescence resonance energy transfer (ECL-RET) system from Eu3 +-doped CdS nanocrystals (CdS:Eu NCs) to Ru(bpy)32 +-doped silica nanoparticle (RuSi) was designed for ultrasensitive detection of prostate-specific antigen (PSA). The platform consists of a CdS:Eu NCs film on glassy carbon electrode (GCE) as ECL emitter and a sandwich-type immunocomplex formed by the primary antibody, PSA and RuSi-tagged second antibody. This system showed a maximum of 4.83-fold enhancement of ECL intensity at 620 nm due to the efficient ECL-RET and high Ru(bpy)32 + quantum yields. By the signal amplification of RuSi and the specific antibody–antigen interactions, this ECL-RET system could sensitively respond down to 1.0 fg mL− 1 PSA.

A Novel Electrochemical Immunosensor for Prostate-Specific Antigen Based on Noncovalent Nanocomposite of Ferrocene Monocarboxylic Acid with Graphene Oxide

A novel electrochemical immunosensor was developed for the determination of prostate-specific antigen based on immobilization of appropriate antibodies on gold nanoparticles and a poly-(2,6-pyridinediamine) modified electrode. The nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide was prepared by a π-π stacking interaction and was used as the electrochemical probe. A sandwich-type complex immunoassay was applied with polyclonal prostate-specific antigen antibodies labeled with the nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide. In order to improve the sensitivity, a potentiostatic method was used to reduce graphene oxide. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the assembly process and the performance of the immunosensor. Under optimal conditions, the peak current of the immunosensor increased with concentration, showing a linear relationship between the peak current and the logarithm of the prostate-specific antigen concentrations in a wide range of 2.0 pg mL−1 to 10.0 ng mL−1 with a low detection limit of 0.5 pg mL−1. The immunosensor was used for the determination of prostate-specific antigen in serum.

Increased expression of GCNT1 is associated with altered O-glycosylation of PSA, PAP, and MUC1 in human prostate cancers.

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues. We interrogated prostate cancer gene expression data for reproducible changes in expression of glycoprotein synthetic enzymes. Over-expression of GCNT1 was validated in prostate samples using RT-PCR. ELISA was used to measure core 2 O-linked glycan sialyl Lewis X (sLe(x) ) of prostate specific antigen (PSA), Mucin1 (MUC1), and prostatic acidic phosphatase (PAP) proteins. A key glycosyltransferase, GCNT1, was consistently over-expressed in several prostate cancer gene expression datasets. RT-PCR confirmed increased transcript levels in cancer samples compared to normal prostate tissue in fresh-frozen prostate tissue samples. ELISA using PSA, PAP, and MUC1 capture antibodies and a specific core 2 O-linked sLe(x) detection antibody demonstrated elevation of this glycan structure in cancer compared to normal tissues for MUC1 (P = 0.01), PSA (P = 0.03) and near significant differences in PAP sLe(x) levels (P = 0.06). MUC1, PSA and PAP protein levels alone were not significantly different between paired normal and malignant prostate samples. GCNT1 is over-expressed in prostate cancer and is associated with higher levels of core 2 O-sLe(x) in PSA, PAP and MUC1 proteins. Alterations of O-linked glycosylation could be important in prostate cancer biology and could provide a new avenue for development of prostate cancer specific glycoprotein biomarkers.

Presence of PSA auto-antibodies in men with prostate abnormalities (prostate cancer/benign prostatic hyperplasia/prostatitis)

Prostate-specific antigen (PSA), produced by the prostate, liquefies post-ejaculate semen. PSA is detected in semen and blood. Increased circulating PSA levels indicate prostate abnormality [prostate cancer (PC), benign prostatic hyperplasia (BPH), prostatitis (PTIS)], with variance among individuals. As the prostate has been proposed as an immune organ, we hypothesise that variation in PSA levels among men may be due to presence of auto-antibodies against PSA. Sera from healthy men (n=28) and men having prostatitis (n=25), BPH (n=30) or PC (n=29) were tested for PSA antibody presence using enzyme-linked immunosorbent assay (ELISA) values converted to standard deviation (SD) units, and Western blotting. Taking ≥2 SD units as cut-off for positive immunoreactivity, 0% of normal men, 0% with prostatitis, 33% with BPH and 3.45% with PC demonstrated PSA antibodies. One-way analysis of variance (anova) performed on the mean absorbance values and SD units of each group showed BPH as significantly different (P<0.01) compared with PC and prostatitis. All others were nonsignificant (P<0.05). Men (33%) with BPH had PSA antibodies by ELISA and Western blot. These discoveries may find clinical application in differential diagnosis among prostate abnormalities, especially differentiating BPH from prostate cancer and prostatitis.

Electrochemical immunoassay for the prostate specific antigen using ceria mesoporous nanospheres

We report on a sensitive electrochemical immunoassay for the prostate specific antigen (PSA). An immunoelectrode was fabricated by coating a glassy carbon electrode with multiwalled carbon nanotubes, poly(dimethyldiallylammonium chloride), CeO2 and PSA antibody (in this order) using the layer-by-layer method. The immunosensor is then placed in a sample solution containing PSA and o-phenylenediamine (OPD). It is found that the CeO2 nanoparticles facilitate the electrochemical oxidation of OPD, and this produces a signal for electrochemical detection of PSA that depends on the concentration of PSA. There is a linear relationship between the decrease in current and the concentration of PSA in the 0.01 to 1,000 pg mL−1 concentration range, and the detection limit is 4 fg mL−1. The assay was successfully applied to the detection of PSA in serum samples. This new differential pulse voltammetric immunoassay is sensitive and acceptably precise, and the fabrication of the electrode is well reproducible.

A nanowire-based label-free immunosensor: Direct incorporation of a PSA antibody in electropolymerized polypyrrole

We have suggested a novel method for the preparation of a label-free electrochemical immunosensor for the detection of prostate-specific antigen (PSA) as target marker for prostate cancer. Direct incorporation of PSA antibody (anti-PSA) into polypyrrole (Ppy) electropolymerized on a three-dimensional Au nanowire array has resulted in enhanced molecular interactions, ultimately leading to improved sensing performance. The electrochemical performance of the nanowire-based immunosensor array were characterized by (1) differential pulse voltammetry (DPV) to evaluate the specific recognition of PSA, (2) impedance and cyclic voltammetry to observe surface resistance and electroactivity, and (3) scanning electron microscopy (SEM) to demonstrate the three-dimensional architecture. The vertically-aligned geometric organization of Ppy provides a novel platform to improve the anti-PSA loading capacity. Overall, enhanced electrochemical performance of the proposed immunosensor has been demonstrated by its linear response over PSA concentrations ranging from 10fgmL−1 to 10ngmL−1 and a detection limit of 0.3fgmL−1, indicating that the strategy proposed here has great potential for clinical applications.

Immune impact induced by PSA-tricom, a therapeutic vaccine for prostate cancer.

245 Background: PSA-TRICOM is a vector-based therapeutic cancer vaccine designed to generate a targeted anti-tumor immune response against prostate-specific antigen (PSA)–expressing tumor cells. Early clinical trials have evaluated the immunologic impact of this vaccine and demonstrated promising clinical activity. PSA-TRICOM is being evaluated in a phase III trial in metastatic castration resistant prostate cancer (mCRPC). Methods: We recently conducted a broad overview of both published and new data which analyzed the immune responses to PSA-TRICOM. Immune responses included ELISPOT for antigen-specific immune response and flow-cytometry analysis of peripheral immune cells. Results: 104 patients (pts) with prostate cancer were tested for T-cell responses and 59 out of 104 (57%) demonstrated a greater than or equal to 2-fold increase in PSA-specific T cells 4 weeks after vaccine. The responders had a median 5-fold increase relative to pre-vaccine levels. For most pts PSA-specific immune responses (likely memory cells) seen 28 days following the most recent vaccine are quantitatively similar to levels of circulating influenza-specific T cells in the same pts. In addition, 19 out of 28 pts (68%) evaluated demonstrated immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). Since PSA-TRICOM is designed to generate a cellular (TH1 immune response), it is not surprising that 2 out of 349 pts (&lt;1.0%) demonstrated evidence of PSA antibody induction following vaccine. This suggests that post-vaccine PSA kinetics were not affected by PSA antibodies. Conclusions: PSA-TRICOM has demonstrated the ability to generate immune responses. Despite these findings, it is important to note that systemic immune response to PSA may underestimate the true therapeutic immune response since it does not measure cells that trafficked to tumor or antigen spreading. Furthermore, while the entire PSA gene is the vaccine, only one epitope of PSA is specifically evaluated in the T-cell responses. Further immune analysis continues in an ongoing phase III of PSA-TRICOM in mCRPC (NCT01322490), accruing worldwide, and two trials combining PSA-TRICOM with enzalutamide (biochemical recurrence/ NCT01875250 and mCRPC/ NCT01867333) currently accruing at NCI. Clinical trial information: multiple trials.

A highly sensitive prostate-specific antigen immunosensor based on gold nanoparticles/PAMAM dendrimer loaded on MWCNTS/chitosan/ionic liquid nanocomposite

We have developed a sensitive electrochemical immunosensor for the detection of prostate-specific antigen (PSA), based on covalently immobilizing of anti-PSA and redox mediator (thionine) onto gold nanoparticles–incorporated polyamidoamine dendrimer (AuNPs–PAMAM) and multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite (MWCNTs/IL/Chit) as the support platform. The MWCNTs/IL/Chit nanocomposite and synthesized AuNPs were characterized using SEM and TEM microscopy techniques. Greatly amplified immunoassay was established by sandwiching the antigen between anti-PSA immobilized on the MWCNTs/IL/Chit/AuNPs–PAMAM interface and anti-PSA labeled with horseradish peroxidase (HRP-labeled anti-PSA) as secondary antibody. Phtaloyl chloride (Ph) was used as linking agent for the subsequent immobilization of AuNPs–PAMAM onto platform and anti-PSA antibody and thionine onto AuNPs–PAMAM dendrimer. The increased electrocatalytic reduction of H2O2 by HRP was monitored by differential pulse voltammetry technique. Under optimized condition the calibration curve for PSA concentration was linear up to 80ngml−1 with detection limit (signal-to-noise ratio of 3) of 1pgml−1. AuNPs–PAMAM dendrimer as platform not only increased the amount of thionine and PSA antibody but also the electron transfer process accelerated by encapsulated AuNPs. Moreover, the proposed PSA immunosensor exhibited excellent stability and reproducibility. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. In addition, impedance technique was used as simple, rapid, low cost label free analytical method for PSA measurement with detection limit of 0.5ngml−1 at concentration range up to 25ngml−1. The results indicate that the present protocol is quite promising in developing other electrochemical immunosensors.

Real-Time and Label-Free Detection of the Prostate-Specific Antigen in Human Serum by a Polycrystalline Silicon Nanowire Field-Effect Transistor Biosensor

In this research, we used a polycrystalline silicon nanowire field-effect transistor (poly-Si NWFET) as a biosensor that employs the sidewall spacer technique instead of an expensive electron beam lithography method. When compared with commercial semiconductor processes, the sidewall spacer technique has the advantages of simplicity and low cost. In this study, we employed a novel poly-Si NWFET device for real-time, label-free, and ultrahigh-sensitivity detection of prostate-specific antigen (PSA) in human serum. Since serum proteome is very complex containing high levels of salts and other interfering compounds, we hereby developed a standard operating procedure for real-sample pretreatment to keep a proper pH value and ionic strength of the desalted serum and also utilized Tween 20 to serve as the passivation agent by surface modification on the NWFET to reduce nonspecific binding for medical diagnostic applications. We first modified 3-aminopropyltriethoxysilane on the surface of a poly-Si nanowire device followed by glutaraldehyde functionalization, and the PSA antibodies were immobilized on the aldehyde terminal. While PSA was prepared in the buffers to maintain an appropriate pH value and ionic strength, the results indicated that the sensor could detect trace PSA at less than 5 fg/mL in a microfluidic channel. The novel poly-Si NWFET is developed as a diagnostic platform for monitoring prostate cancer and predicting the risk of early biochemical relapse.

Highly sensitive immunosensing of prostate-specific antigen based on ionic liquid–carbon nanotubes modified electrode: Application as cancer biomarker for prostatebiopsies

A novel and highly sensitive electrochemical immunosensor was developed for detection of prostate specific antigen (PSA) based on immobilization of PSA-antibody (anti-PSA) onto robust nano composite containing multiwalled carbon nanotubes (MWCNTs) and ionic liquid (IL)1-buthyl-methylpyrolydinium bis (trifluromethyl sulfonyl) imide [C4mpyr][NTf2]. Thionine was used as redox system for electrochemical probe. The adsorbed thionine displayed a surface-controlled electrode process with electron transfer rate constant of 6.5s−1 and surface coverage of 3.1×10−11molcm−2. The PSA antibody (anti-PSA) was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti-PSA labeled with horseradish peroxidase (HRP) as secondary antibody and H2O2 as the substrate. Under optimal conditions, the DPV peak current of the immunosensor increased linearly with increasing PSA concentration in two concentration ranges, 0.2–1.0 and 1–40ngml−1 with detection limit 20pgml−1. The intra-assay and inter-assay coefficients of variation (CVs) for five replicate determinations were 2% and 1.35% at 5ngml−1 PSA. The immunosensor has been used for PSA detection in serum samples and the accuracy of the method was demonstrated by comparison to the standard ELISA assay. Furthermore, the immunosensor can be used for detection of PSA in prostate tissue samples. The simple one-step fabrication method, high sensitivity, good reproducibility and repeatability as well as acceptable accuracy for PSA detection in human serum samples are the main advantages of this immunosensor. Furthermore, the results indicate that differences in PSA concentration can be detected with the proposed immunosensor for cancer tissue samples which holds great promise in clinical screening of cancer biomarkers.

Abstract 1268: Accurate measurement of serum total and free PSA using immunoaffinity depletion coupled to SRM: Correlation with clinical immunoassays

Abstract Accurate and reproducible quantification of proteins at the sub-ng/mL concentration range is required for the application of protein biomarkers to clinical assays using blood plasma or serum. The standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention is sandwich immunoassay; although highly sensitive, the development of a specific immunoassay is time-consuming and associated with high costs due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically, selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissues and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed high levels of correlation (R2 values ranging from 0.90 to 0.99) in independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. In addition, we developed an ultrasensitive antibody-free strategy for achieving accurate detection and quantification of proteins at ∼50 pg/mL level in human plasma/serum using high resolution reversed phase separations for target enrichment along with intelligent selection of target fractions via on-line SRM monitoring of internal standards and fraction multiplexing prior to SRM quantification. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, the simultaneous measurement of the free and bound forms of PSA, as well as many other biomarkers that have similar complexation/binding characteristics, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS. This work represents a major technological advance for achieving pg/mL levels of protein quantification in blood plasma without the need for specific antibodies for enrichment and holds great promise for broad applications in biomarker verification and systems biology applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1268. doi:1538-7445.AM2012-1268