Quantification Of Prostate Specific Antigen Research Articles

Prostate-specific antigen levels in patients with risk factors for prostate carcinoma disorders

ABSTRACT Objective: To verify the association between the prostate-specific antigen (PSA) serum concentration and the presence of risk factors for prostate diseases in adult patients with urogenital infections. Materials and methods: Analytical cross-sectional study of PSA in 60 patients aged 40 to 65 years, from January to December 2013. PSA quantification was performed in serum by solid-phase chemiluminescence in two label cycles: 1. mouse monoclonal antibody (mAb); and 2. goat polyclonal antibody (pAbs). After each cycle the free antigen was removed. The readings were taken on the INMULITE ONE Siemens® automated equipment. From the PSA, 0.0-2.5 ng/ml was used to calculate the antigen distribution trend, and a bivariate statistical analysis of PSA was as opposed to previous exams, and endogenous and environmental risk factors detected. Results: Patients between 50-60 years of age, with a family history of urogenital and sexually transmitted infections, toxic habits, and exposure to occupational risk prevailed. Sixty-two percent of patients presented normal PSA levels, and the remaining presented slightly elevated and very high PSA levels. In the statistical analysis, a significant association (p < 0.001) of PSA was found as opposed to previous tests. A significant association (p = 0.001) was also found between PSA versus age, family history, personal pathological history, toxic habits, and risk exposure. Conclusion: The high association rate found between PSA versus age and other risk factors could be used as a predictive value for prostate cancer (Pca) or other prostate disorders.

Dual-Mode Electrochemical Assay of Prostate-Specific Antigen Based on Antifouling Peptides Functionalized with Electrochemical Probes and Internal References.

Sensitive and selective detection of target analytes in complex biological samples is currently a major challenge. Herein we constructed a dual-mode antifouling electrochemical sensing platform for the detection of prostate-specific antigen (PSA) based on two kinds of antifouling peptides functionalized with a graphene oxide-Fe3O4-thionine (GO-Fe3O4-Thi) probe and internal reference ferrocene (Fc), respectively. The longer peptide (Pep1) modified with the GO-Fe3O4-Thi probe was designed to contain a peptide sequence (HSSKLQK) capable of being recognized and cut by PSA. The GO-Fe3O4-Thi probe functions not only as a peroxidase mimick (GO-Fe3O4) but also works as an electrochemical probe due to the presence of thionine (Thi). The concentration of PSA can be measured through both the increase of differential pulse voltammetry (DPV) signal change of Thi and the decrease of chronoamperometry (CA) signal of the reduction of H2O2 electrocatalyzed by GO-Fe3O4. The shorter peptide (Pep2) was tagged with Fc, whose DPV signal remained constant and was independent of the presence of PSA, and it was used as an internal reference to ensure the reliability and accuracy of the measurement. The dual-mode PSA sensor exhibits a wide linear range from 5 pg/mL to 10 ng/mL, with low detection limits of 0.76 and 0.42 pg/mL through DPV and CA modes, respectively. More importantly, owing to the antifouling capability of the designed peptides, the biosensor performances remained operable even in human serum, indicating feasibility of the electrochemical biosensor for practical PSA quantification in complex samples.

Highly sensitive voltammetric immunosensor for the detection of prostate specific antigen based on silver nanoprobe assisted graphene oxide modified screen printed carbon electrode

In this work, we developed an effective voltammetric immunosensing platform for the sensitive detection of prostate specific antigen (PSA) utilizing a graphene oxide (GO) modified screen-printed carbon electrode (SPCE) hybridized with the ex-situ prepared silver nanoparticles (AgNPs) as a probe and signal transducer. The sensing platform comprises a direct-type immunoassay involving the selective interaction of PSA with anti-PSA. The surface morphology and analytical performance of the modified SPCE were characterized through relevant instrumentations. The changes in the voltammetric reduction current of AgNPs at 0.11 V in the sensor electrode was correlated to the PSA concentration. Under optimum conditions, the fabricated immunosensor exhibited a sensitive response to PSA with a limit of detection (LOD) of 0.27 ng mL−1 and a dynamic calibration range of 0.75–100.0 ng mL−1. We demonstrated that the participation of AgNPs along with GO modification contribute to the desired signal amplification and sensitive detection of PSA. It is anticipated that the proposed immunosensor can serve as a biomarker and potentially be utilized for the real sample quantification of PSA.

Carbon Nanotubes Modified With Au for Electrochemical Detection of Prostate Specific Antigen: Effect of Au Nanoparticle Size Distribution.

Different functionalized Multi-Wall Carbon Nanotube and gold nanoparticles (AuNPs) were synthesized as biosensor electrodes. These materials have been applied to the detection of the Prostate Specific Antigen (PSA). The synthesis of AuNPs was carried out using polyvinylpyrrolidone (PVP) as protecting agent. The PVP/Au molar ratio (0.5 and 50) controls the nanoparticle size distribution, obtaining a wide and narrow distribution with an average diameter of 9.5 and 6.6 nm, respectively. Nanoparticle size distribution shows an important effect in the electrochemical performance of the biosensor, increasing the electrochemical active surface area (EASA) and promoting the electron-transfer from the redox probe (Ferrocene/Ferrocenium) to the electrode. Furthermore, a narrow and small nanoparticle size distribution enhances the amount of antibodies immobilized on the transducer material and the performance during the detection of the PSA. Significant results were obtained for the quantification of PSA, with a limit of detection of 1 ng·ml−1 and sensitivities of 0.085 and 0.056 μA·mL·ng−1 for the two transducer materials in only 5 min of detection.

Fluorometric nanoprobes for simultaneous aptamer-baseddetection of carcinoembryonic antigen and prostate specific antigen.

A "turn-on" fluorometric assay based on the combined effects of fluorescence resonance energy transfer (FRET) and internal filter effect (IFE) is described for the rapid and ultrasensitive detection of both carcinoembryonic antigen (CEA) and prostate specific antigen (PSA). Their unique porous structures and high specific surface enable mesoporous silica nanoparticles (MSNs) to load a large number of CdTequantum dots (QDs). These amplify the fluorescence signal and provide a platform to fabricate more distinctly fluorescent MSNs (QD-MSNs). Two kinds of QD-MSNs with the maximum emission wavelengths at 590nm (orange)and 731nm (dark red)were fabricated and served as two types of fluorescent probes for the dual detection. Two aptamers were covalently connected to fluorescent MSNs as the recognition unit to warrant the selectivity of assay. The fluorescence of QD-MSNs can be quenched by molybdenum disulfide nanosheets (MoS2) due to FRET mechanism, IFE also contributed to thethe reduction of fluorescence intensity. The fluorescence of QD-MSNs was further recovered in the presence of CEA and PSA attributing to the excellent specificity of aptamers. A "turn-on" fluorescent two-channel nanoprobe is introduced for simultaneous quantification of CEA and PSA. The respective limits of detection (at S/N= 3) are 0.7fg•mL-1 for CEA and 0.9fg•mL-1 for PSA. Graphical abstract Schematic presentation of theturn-on fluorescent nanoprobes for simultaneous detection of CEA and PSA.

Sandwich pair nanobodies, a potential tool for electrochemical immunosensing serum prostate-specific antigen with preferable specificity

Prostate-Specific Antigen (PSA) is a crucial biomarker for screening prostate cancer, but a sensitive and selective immunosensor for rapid quantification of serum PSA remains to be developed. In this study, a sandwich pair of nanobodies (Nbs) (i.e., Nb2 and Nb40) against PSA surface antigen was obtained from an alpaca-derived immune phage display library. A sandwich-type immunosensor for the sensitive and selective detection of PSA in serum samples was ingeniously designed based on the pair of Nbs. The small size of Nb40 allowed high capture densities on the surface of reduced graphene oxide (rGO) nanocomposed with massive Au nanoparticles (rGO@AuNPs), which significantly improved the conductivity and provided a large area to anchor many primary antibodies. The secondary antibody Nb2 fused with streptavidin -binding peptide (SBP) cooperated with Nb40 for PSA sandwiching. Accompanying introduction of horseradish peroxidase-streptavidin (HRP-SA) coupled with Nb2-SBP, the faradaic current was linearly correlated with the logarithm of PSA concentration in a range of 0.1–100 ng mL−1. More importantly, this immunosensor exhibited excellent selectivity, stability, and reproducibility due to the sandwich pair Nbs. The proposed immunosensor was successfully applied in determining PSA in serum samples and could be used for the sensitive and specific detection of PSA.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

An endoglycosidase-assisted LC-MS/MS-based strategy for the analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using prostate-specific antigen (PSA) as example

Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60 ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1 ng/ml and total PSA lower than 0.5 ng/ml. The imprecision of the method over two days ranged from 9.7–23.2% for core-fucosylated PSA and 10.3–18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers (“marker refinement”) in cancer research.

Detection of prostate specific antigen (PSA) in human saliva using an ultra-sensitive nanocomposite of graphene nanoplatelets with diblock-co-polymers and Au electrodes.

Prostate-specific antigen (PSA) is a commonly used biomarker for the detection of prostate cancer (PCa) and there are numerous data available for its invasive detection in the serum and whole blood. In this work, an electrochemical sensing method was devised to detect traces of PSA in human saliva using a hybrid nanocomposite of graphene nanoplatelets with diblock co-polymers and Au electrodes (GRP-PS67-b-PAA27-Au). The pure graphitic composition on filter paper provides significantly high electrical and thermal conductivity while PS67-b-PAA27 makes an amphiphilic bridge between GRP units. The sensor utilizes the binding of an anti-PSA antibody with an antigen-PSA to act as a resistor in a circuit providing an impedance change that in turn allows for the detection and quantification of PSA in saliva samples. A miniaturized electrical impedance analyzer was interfaced with a sensor chip and the data were recorded in real-time using a Bluetooth-enabled module. This fully integrated and optimized sensing device exhibited a wide PSA range of detection from 0.1 pg mL-1 to 100 ng mL-1 (R2 = 0.963) with a lower limit of detection of 40 fg mL-1. The performance of the biosensor chip was validated with an enzyme-linked immunosorbent assay technique with a regression coefficient as high as 0.940. The advantages of the newly developed saliva-PSA electrical biosensor over previously reported serum-PSA electrochemical biosensors include a faster response time (3-5 min) to achieve a stable electrical signal for PSA detection, high selectivity, improved sensitivity, no additional requirement of a redox electrolyte for electron exchange and excellent shelf life. The presented sensor is aimed for clinical commercialization to detect PSA in human saliva.

Development of immunomagnetic droplet-based digital immuno-PCR for the quantification of prostate specific antigen

Immuno-PCR (IPCR) is an immunoassay that employs DNA as the signal generator and utilizes both the versatility of antigen–antibody interactions and the exponential amplification power of PCR.

Heparin‐gold Nanoparticles and Liquid Crystal Applied in Label‐free Electrochemical Immunosensor for Prostate‐specific Antigen

AbstractA label‐free electrochemical immunosensor based on the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br−Py), together with heparin‐stabilized gold nanoparticles (AuNP‐Hep) and Nafion is proposed for the determination of prostate‐specific antigen (PSA). The Br−Py liquid crystal presented redox properties and good film‐forming abilities on the electrode surface, and thus it is a suitable alternative as a redox probe for a label‐free electrochemical immunosensor, which could simplify the analysis methodology. The stepwise construction of the immunosensor and the incubation process (immunocomplex formation) were characterized by voltammetry and electrochemical impedance spectroscopy. The proposed immunosensor could directly detect PSA concentrations in the incubation samples, based on the suppression of the Br−Py redox peak (‘base peak’) current. After optimization, the immunosensor exhibited a linear response to PSA concentrations in the range of 0.1 to 50 ng mL−1, with a calculated detection limit of 0.08 ng mL−1. The reproducibility (coefficient of variance less than 3.0 %), selectivity and accuracy of the methodology were adequate. The immunosensor was satisfactorily applied in the quantification of PSA in human blood plasma samples.

Sensitive prostate specific antigen quantification using dihydrolipoic acid surface-functionalized phosphorescent quantum dots

Herein, high-quality Mn-doped ZnS quantum dots (QDs) have been synthesized using a facile approach directly in aqueous media. The surface of the obtained QDs was further modified by cap-exchange of the native cysteine shell with dihydrolipoic acid (DHLA) ligands resulting in nanocrystals with high water-stability having an intense phosphorescent signal. Covalent bioconjugation of the DHLA-coated nanoparticles with an anti-IgG antibody was then carried out. Interestingly the QD immunoprobe (QD-labelled antibodies) maintained an intense phosphorescence emission, without any significant spectral-shift (as compared to the free QDs). Coupling of an asymmetric flow field flow fractionation technique to an elemental mass spectrometry detection enabled the accurate determination of the efficiency of the bioconjugation reaction.The obtained nanoparticle-antibody bioconjugate was then applied to develop a quantitative sandwich-type phosphorescent immunoassay for Prostate Specific Antigen (PSA), and a limit of detection (LOD) of 17 pg mL−1 of PSA was achieved and allow to quantify such biomarker in samples within clinically relevant levels. Finally, the assay was validated for the quantification of PSA in the cellular media of prostate cancer cells. Obtained results proved the robustness of the proposed immunoassay based on long-lived phosphorescence measurements against eventual photoluminescent interferences significantly affecting the conventional short-lived fluorescence detection.

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 0.25-10ng/mL. Based on a S/N of 3, the detection limit for PAS was estimated to be 0.092ng/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.

Electrochemical sandwich immunoassay for the prostate specific antigen using a polyclonal antibody conjugated to thionine and horseradish peroxidase

The authors present a voltammetric immunoassay for the quantification of the prostate specific antigen (PSA), a well-established tumor marker. Biotinylated anti- PSA antibodies were immobilized on the surface of a nano-TiO2-modified carbon paste electrode using specific interaction between streptavidin and biotin. Streptavidin itself was covalently grafted to the electrode surface via 4-aminobenzoic acid film. Signal transduction was then performed using polyclonal antibodies conjugated with horseradish peroxidase (HRP) and thionine. An amplified catalytic reduction current was observed in the presence of H2O2 because more than one polyclonal antibody linked to each antigen. Each step of the preparation of the immunosensor was monitored via electrochemical impedance spectroscopy. The electrode, if operated at a typical potential of 0.3 V vs. Ag/AgCl and using hexacyanoferrate as an electrochemical probe, exhibits linear responses in the 0.10 to 5.0 ng·mL−1 and 5.0 to 100 ng·mL−1 PSA concentration ranges, with a detection limit of 200 pg·mL−1. The accuracy of the biosensor was confirmed by analyzing a certified human serum sample, and this indicated that the immunosensor is well suited for the quantification of PSA.

Amplified using DNase I and aptamer/graphene oxide for sensing prostate specific antigen in human serum

In this paper, we proposed an amplified assay using DNase I for sensitively sensing prostate specific antigen (PSA) based on CdSe/ZnS quantum dot labelled PSA aptamer/graphene oxide (QD-aptamer/GO). In this sensing system, GO could strongly bind QD conjugated aptamer and quench the fluorescence of QD, furthermore, GO could protect DNA from nuclease cleavage. When binding target PSA, aptamer probe released from the surface of GO nanosheet, then the free aptamer could be cleaved by nuclease, thus the QD and PSA were liberated, after that, the released PSA would bind another aptamer on GO nanosheet and start a new cycle, which resulted in amplification of restoring signal. The probe possessed steady fluorescence signal and high specificity due to the anti photobleaching of QD and the high affinity of aptamer to PSA. Under the optimum experimental conditions, fluorescence intensity increased linearly with the PSA concentration between 0.1fgmL−1 and 3fgmL−1 with the limit of quantification of 0.05fgmL−1, which was three orders of magnitude lower than that without DNase I. Finally, the method was applied to quantification of PSA in human serum samples.

Direct sensing of cancer biomarkers in clinical samples with a designed nanopore.

Here, we show a designed solid-state nanopore sensor for the direct sensing and quantification of prostate-specific antigen (PSA) as cancer biomarker in serum without any pretreatment. This nanopore technique provides a convenient, fast, and low-cost quantification of cancer biomarkers in clinical samples.

Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions.

Sensitive detection and glycoprofiling of a prostate specific antigen using impedimetric assays.

This study presents a proof-of-concept for the development of an impedimetric biosensor for ultra-sensitive glycoprofiling of prostate specific antigen (PSA). The biosensor exhibits three unique characteristics: (1) analysis of PSA with limit of detection (LOD) down to 4 aM; (2) analysis of the glycan part of PSA with LOD down to 4 aM level and; (3) both assays (i.e., PSA quantification and PSA glycoprofiling) can be performed on the same interface due to label-free analysis.

Testing the variability of PSA expression by different human prostate cancer cell lines by means of a new potentiometric device employing molecularly antibody assembled on graphene surface

Prostate Specific Antigen (PSA) is widely used as a biomarker for prostate cancer. Recently, an electrochemical biosensor for PSA detection by means of molecularly imprinted polymers (MIPs) was developed. This work evaluated the performance and the effectiveness of that PSA biosensor in screening the biomarker PSA in biological media with complex composition, collected from different human prostate cell line cultures. For that, the prostate cancer LNCaP and PC3 cells, and the non-cancerous prostate cell line PNT2 were cultured for 2, 7 and 14days in either α-MEM or RPMI in the presence of 10% or 30% fetal bovine serum. Human gingival fibroblasts were used as a non-cancerous non-prostatic control. The different culture conditions modulated cellular proliferation and the expression of several prostate markers, including PSA. The electrochemical biosensor was able to specifically detect PSA in the culture media and values obtained were similar to those achieved by a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit, the most commonly used method for PSA quantification in prostate cancer diagnosis. Thus, the tested biosensor may represent a useful alternative as a diagnostic tool for PSA determination in biological samples.

Electrical nanowell diagnostics sensors for rapid and ultrasensitive detection of prostate-specific antigen.

Alumina nanowell based disposable diagnostic biosensor for detecting and quantifying levels of prostate-specific antigen (PSA) from human serum has been designed, fabricated and tested. The biosensors were designed by integrating nanoporous alumina membranes onto printed circuit board platforms, resulting in the generation of high-density nanowell arrays with gold base electrodes. The size and density of the nanowells were leveraged toward achieving sieving action for size-based exclusion of nonspecific molecules and size-based confinement of the target PSA molecules. We demonstrated PSA detection between 0.01 and 1000 ng/ml and detection and quantification of PSA from a 17 patient cohort validated using the Beckman Access system with >95% correlation.