Label electrochemical immunosensor for prostate-specific antigen based on graphene and silver hybridized mesoporous silica

Prostate-specific antigen (PSA), as the specificity of prostate cancer markers, has been widely used in prostate cancer diagnosis and screening. In this study, we fabricated an electrochemical immunosensor for PSA detection using the amino-functionalized graphene sheet–ferrocenecarboxaldehyde composite materials (NH₂-GS@FCA) and silver hybridized mesoporous silica nanoparticles (Ag@NH₂-MCM48). Under optimal conditions, the fabricated immunosensor showed a wide linear range with PSA concentration (0.01–10.0 ng·ml⁻¹). Low detection limit (2 pg·ml⁻¹) proved the high sensitivity. In addition, the immunosensor possessed good stability and reproducibility. Moreover, the application to PSA analysis in serum samples yielded satisfactory results.     

Development of Metal-Enhanced Fluorescence-Based Aptasensor for Thrombin Detection Using Silver Dendritic Nanostructures

Metal-enhanced fluorescence (MEF) phenomenon has shown a promising potential in the field of fluorescence-based biological sensing. In this study, we optimized the electroless metal deposition method to fabricate silver dendritic nanostructures as effective MEF active substrates. Then, an aptasensor was developed for thrombin detection using the established surfaces. For this purpose, thiolated 29-mer thrombin-binding aptamers (TBA₂₉ (12T) SH) as capturing aptamer were immobilized on the surface of silver dendritic nanostructures, then thrombin was sandwiched between the capturing aptamer and Cy5-labeled 15-mer thrombin aptamer (TBA_15-Cy5). Quantitative analysis was performed through fluorescence signal measurement. The established aptasensor presented satisfactory sensitivity and selectivity and exhibited a limit of detection (LOD) as low as 32 pM. This aptasensor was also able to detect thrombin in the human serum at picomolar levels. Furthermore, the ease and relatively low-cost of fabrication of this platform introduce it as a tool with great potential for the clinical diagnosis of diseases and also for improving sensitivity of a variety of technologies which exploit fluorescent dyes for analyte detection, at ultra-trace levels, in complex matrices.

     

A graphene oxide-peptide fluorescence sensor for proteolytically active prostate-specific antigen

This paper presents a novel sensor to detect proteolytically active prostate-specific antigen (PSA) by assembling a purpose-designed FITC-labeled peptide with graphene oxide (GO). The fluorescence of the dye-labeled peptide was quenched in the presence of GO. Reaction of the sensor with PSA cleaves the peptide, leading to the release of the dye moiety and a great increase in fluorescence intensity in a dose- and time-dependent manner, and PSA can be quantified accordingly. This approach is simple compared to existing methods since the GO-peptide-based sensor is easily assembled and detection can be achieved without the involvement of complicated procedures. Moreover, the applicability of the method has been demonstrated by detecting PSA in spiked urine samples.     

General and hybrid correlation nuclear magnetic resonance analysis of phosphorus in Phytophthora palmivora

As a specific tumor marker, prostate-specific antigen (PSA) is widely used for the early diagnosis of prostate cancer. Sensitive and specific methods are required to improve the diagnostic accuracy of PSA detection. In the current study, we compared the immuno-polymerase chain reaction (immuno-PCR) method with the solid-phase proximity ligation assay (SP-PLA) with respect to the detection of PSA. Using oligonucleotide-labeled antibody probes, we used both immuno-PCR and SP-PLA to detect trace levels of PSA. The nucleic acid sequences can be monitored using real-time PCR. SP-PLA, however, was found to be superior in terms of both the detection limit and the dynamic range. To detect even lower levels of PSA, we used the loop-mediated isothermal amplification (LAMP) method to measure the levels of reporter DNA molecules in SP-PLA. The sensitivity of the LAMP method is 0.001 pM, which is approximately 100-fold higher than the sensitivities of the other assays. The results suggest that an SP-PLA- and LAMP-based protocol with oligonucleotide-labeled antibody probes may have great application in detecting PSA or other proteins present at trace levels.     

Silicon Nitride-BP-Based Surface Plasmon Resonance Highly Sensitive Biosensor for Virus SARS-CoV-2 Detection

In this study, we propose a surface plasmon resonance (SPR)-based biosensor using silicon nitride (Si₃N₄), black phosphorous (BP), and thiol-tethered DNA as a ligand for fast detection of the SARS-CoV-2 virus. In the proposed biosensor, we have deposited silver (Ag), Si₃N₄, and BP on the base of the BK-7 prism and investigated the performance parameters on the probe in different combinations of the mentioned materials. Herein, three (Ag, Si₃N₄, and BP) different configurations are introduced and compared for the detection of SARS-CoV-2. Furthermore, with the help of the transfer matrix method (TMM), all the three configurations have been analyzed. Notably, the combination of Ag, Si₃N₄, and BP shows better sensitivity (154°/RIU) when compared with other configurations for the detection of SARS-CoV-2. This work may facilitate a new sensing device to detect SARS-CoV-2, based on the hybrid materials.

     

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.     

Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-specific antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) technology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘RLSSYYSGAG’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn’t show enzymatic activity toward the biosensor in serum solution, likely reflecting the existence of other inhibitory factors besides Zn2+. By expressing the biosensor on cell plasma membrane, the FRET responses were significant, but couldn’t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insufficient speci- ficity to PSA. The biosensor with the previously known ‘HSSKLQ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.     

Biomarkers for prostate cancer

The detection of prostate cancer using a blood test has by many standards changed the face of the disease. Despite this tremendous success, there are limitations attributed to the use of prostate specific antigen (PSA) as a means to screen and detect prostate cancer. PSA, as its name implies, is not specific for prostate cancer and as such is often found elevated in other prostatic diseases/symptoms associated with the aging male. Clearly, more specific marker(s) that could identify which individuals actually have prostate cancer and differentiate them from those without the disease would be of tremendous value. The search for more accurate and clinically useful biomarkers of prostate cancer has been extensive. This has focused on individual markers, as well as groups of markers. Included among these are PSA isoforms, pathological indicators and stains, nucleic acids and others. This article highlights the discovery of PSA as a first blood‐based biomarker for prostate cancer detection, as well as other molecular biomarkers and their potential application in detection of the disease. J. Cell. Biochem. 108: 3–9, 2009. © 2009 Wiley‐Liss, Inc.     

Ultra-High-Sensitive Sensor Based on Surface Plasmon Resonance Structure Having Si and Graphene Layers for the Detection of Chikungunya Virus

Chikungunya virus has been discovered in about 60 countries of the world. It leads to joint pain, joint swelling, headache, muscle pain, and fatigue of the human body. In this work, a surface plasmon resonance (SPR)based sensor is developed to detect chikungunya virus through normal and infected platelets and plasma blood cells. The proposed SPR-based sensor uses silicon and graphene layers coated over the base of a glass prism sputtered with a silver layer. The graphene layer has the advantage of enhancing the biomolecules adsorption on the metal layer. The silicon layer between silver and graphene enhances the sensor performance. The number of graphene layers along with the thicknesses of silicon and silver layers is optimized to get the highest sensitivity of the detector. To investigate the effect of the light source wavelength, simulations are performed for four different wavelengths. The highest sensitivities exhibited by the SPR-based sensor are 393 and 160 deg/RIU for the platelets and plasma cells, respectively.

     

Potentiometric Stripping analysis—a new technique for tissue analysis used to correlate chemical speciation with progress of wound healing

ABSTRACT

PSA has been adapted to the analysis of 20 mg samples of skin for copper at 12 and zinc at 33 ppb of dry mass. This technique can be applied to other metal analyses, can be extended to human samples and produces data which, used as input, will permit considerably more precise chemical speciation data reflecting bioavailabilities etc. Other methods of analysing such low levels are reviewed and all suffer from disadvantages compared with PSA when used in a hospital environment—extremely expensive, highly skilled technologists required, slow turn around of results, large sample volumes necessary, non-applicable to physiological concentrations. The PSA method can now be applied to assessing and monitoring treatment of copper and zinc levels for patients.     

A Highly Sensitive Dual-Core Photonic Crystal Fiber Based on a Surface Plasmon Resonance Biosensor with Silver-Graphene Layer

A highly sensitive dual-core photonic crystal fiber based on a surface plasmon resonance (PCF-SPR) biosensor with a silver-graphene layer is described. The silver layer with a graphene coating not only prevents oxidation of the silver layer but also can improve the silver sensing performance due to the large surface-to-volume ratio of graphene. The dual-core PCF-SPR biosensor is numerically analyzed by the finite-element method (FEM). An average spectral sensitivity of 4350 nm/refractive index unit (RIU) in the sensing range between 1.39 and 1.42 and maximum spectral sensitivity of 10,000 nm/RIU in the sensing range between 1.43 and 1.46 are obtained, corresponding to a high resolution of 1 × 10⁻⁶ RIU as a biosensor. Our analysis shows that the optical spectra of the PCF-SPR biosensor can be optimized by varying the structural parameters of the structure, suggesting promising applications in biological and biochemical detection.

     

Effect of Plasmonic Silver Nanoparticles’ Size on Photophysical Characteristics of 4-Aryloxymethyl Coumarins

The effect of plasmonic silver nanoparticles’ size on photophysical characteristics of four biologically active 4-aryloxymethyl coumarins 4-p-tolyloxymethylbenzo[h] coumarin (4PTMBC), 1-(4-iodo phenoxymethyl)-benzo [f] coumarin (1IPMBC), 4-(4-iodo-phenoxymethyl)-benzo [h] coumarin (4IPMBC), and 4-(4-iodo-phenoxymethyl)- 6-methoxy coumarin (4IPMMC) has been studied using absorption and fluorescence spectroscopy. The size of silver nanoparticles has been estimated by field effect scanning electron microscope technique. The absorption maxima of silver nanoparticles are red shifted with increase in their size. The absorption spectral changes of investigated coumarins with the addition of silver nanoparticles of different sizes suggest their possible interaction with silver nanoparticles. Fluorescence quenching has been observed for all the coumarins with the addition of silver nanoparticles of different sizes. The Stern-Volmer (S-V) plots of fluorescence quenching are found to be linear. The magnitude of quenching rate parameter suggests the involvement of static quenching mechanism. Fluorescence data has been used to estimate binding constants and the number of binding sites. The contribution of diffusion and electron transfer processes in fluorescence quenching mechanism has also been discussed. The values of S-V constant and quenching rate parameter are found to decrease with increase in size of silver nanoparticles.

     

Label-free electrochemical immunosensor based on graphene/methylene blue nanocomposite

For the specificity of prostate cancer markers, prostate specific antigen (PSA) has been widely used in prostate cancer screening, diagnosis, and treatment after monitoring. In normal male serum, PSA can only be detected in traces of 0-4 ng mL(-1). In this paper, we constructed an electrochemical immunosensor for PSA detection using a nanocomposite film of graphene sheets-methylene blue-chitosan (GS-MB-CS) as electrode material. The nanocomposite film showed high binding affinity to the electrode and was used to immobilize the antibody of PSA. The modification procedure was monitored by cyclic voltammetry (CV). An amperometric biosensor was easily developed based on the response of peak current to the capture of PSA induced by specific antigen-antibody reactions. Under optimum conditions, the amperometric signal decreased linearly with PSA concentration (0.05-5.00 ng mL(-1)). A low limit of detection (13 pg mL(-1)) and a high selectivity are obtained. Moreover, the prepared immunosensor was applied for the analysis of PSA in serum samples with satisfactory results. The proposed method may have a promising future in biochemical assays for high selectivity, good reproducibility, and stability.     

Use of 5-Nitroindole-2′-deoxyribose-5′-triphosphate for Labelling and Detection of Oligonucleotides†

Abstract

The 5′-triphosphate of 5-nitroindole-2′-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.

     

经直肠剪切波弹性成像技术联合血清CEA、PSA、FPSA对前列腺良恶性病变的鉴别诊断价值研究

摘要 目的：研究经直肠剪切波弹性成像技术（TRSWE）联合血清癌胚抗原（CEA）、前列腺特异性抗原（PSA）、游离前列腺特异性抗原（FPSA）对前列腺良恶性病变的鉴别诊断价值。方法：选取合肥市第二人民医院2019年1月～2022年6月收治的90例前列腺病变患者,根据病理检查分为前列腺癌组（47例）和前列腺良性病变组（43例）。对所有前列腺病变患者均行TRSWE检查,分析前列腺良恶性病变的图像差异以及弹性模量最大值（Emax）、弹性模量平均值（Emean）。检测所有前列腺病变患者的血清CEA、PSA、FPSA水平并进行对比。采用受试者工作特征（ROC）曲线分析明确Emax值、Emean值以及血清CEA、PSA、FPSA水平联合诊断前列腺良恶性病变的效能。结果：前列腺癌组Emax值、Emean值均高于前列腺良性病变组（均P＜0.05）。前列腺癌组血清CEA、PSA及FPSA水平均高于前列腺良性病变组（均P＜0.05）。经ROC曲线分析发现,Emax值、Emean值以及血清CEA、PSA、FPSA水平联合检测诊断前列腺良恶性病变的效能优于上述5项指标单独检测。结论：TRSWE联合血清CEA、PSA、FPSA对前列腺良恶性病变的鉴别诊断价值较高,可有效提升前列腺癌的检出率,可能值得临床推广应用。     

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1–0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.     

Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels

Variations of the ultrasensitive silver staining method of B. R. Oakley, D. R. Kirsch, and N. R. Morris (Anal. Biochem.105, 361–363 (1980)) have been tested. It was established that the reliability of the method was greatly improved if (i) free silver was carefully washed out before reduction with formaldehyde; (ii) the extent of development was controlled by using methylamine to inactivate the formaldehyde; and (iii) the optimum quantity of ammonia, which was found to be 4 mol/mol of silver was used (this quantity was defined as that which titrates a particular amount of 1 n HCl). The time of preparation of the formaldehyde reducer was found not to be highly critical. In our hands the method can detect down to 0.1 ng of protein/5-mm slot.     

Comparison of quantum dot immunofluorescence histochemistry with conventional immunohistochemistry in detecting Helicobacter pylori infection in paraffin-embedded tissues of gastric biopsy

Quantum dots (QDs) are a new type of fluorescent label, which has been widely used in many biological and biomedical imaging applications. In this study, we used QDs-based immunofluorescence histochemistry (QDs-IHC) and conventional immunohistochemistry (IHC) techniques to perform a retrospective analysis on paraffin-embedded tissues of gastric biopsies in 203 patients (112 of which were HP positive and 91 were negative). The ability of QDs-IHC to detect Helicobacter pylori (HP) in gastric biopsies compared to IHC technology was evaluated. In our study, both methods showed consistent HP morphology and localization. The positive detection rate of HP for QDs-IHC in formalin-fixed and paraffin-embedded (FFPE) tissue was 54.7% (111/203), and the sensitivity and specificity reached 99.11% and 100%, respectively. However the positive detection rate of HP for IHC was 53.7% (109/203), with a sensitivity and specificity of 97.32% and 100%, respectively. Weak positives (1+) were detected in 2 case of QDs-IHC with negative in IHC, and moderate positives (2+) were detected in 3 case of QDs-IHC with weak positives (1+) in IHC. The consistency test showed that the two methods showed good agreement (κ?=?0.980, P?=?0.014), but the sensitivity of QDs-IHC was slightly higher than that of conventional IHC. Our results show that QDs-IHC has strong sensitivity and high specificity. It is superior to conventional IHC in detecting HP infection in FFPE tissues of gastric biopsy, especially in tissues with low HP content.

     

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

     

Carbon nanotube-based ultrasensitive multiplexing electrochemical immunosensor for cancer biomarkers

A multiplexing electrochemical immunosensor was developed for ultrasensitive detection of cancer related protein biomarkers. We employed disposable screen-printed carbon electrode (SPCE) array as the detection platform. A universal multi-labeled nanoprobe was developed by loading HRP and goat-anti-rabbit IgG (secondary antibody, Ab₂) onto multiwalled carbon nanotube (MWNT). This universal nanoprobe was available for virtually any sandwich-based antigen detection and showed superiority in several areas. By using the SPCE array and the universal nanoprobe, we could detect as low as 5 pg mL⁻¹ of prostate specific antigen (PSA) and 8 pg mL⁻¹ of Interleukin 8 (IL-8) with the electrochemical immunosensor. We also demonstrated simultaneous detection of two protein biomarkers with this platform. With these attracted features, our immunoassay system shows promising applications for in-field and point-of-care test in clinical diagnostics.