Evaluation of blood and urine based biomarkers for detection of clinically-significant prostate cancer.

BACKGROUND: Recognizing the limitations of prostate-specific antigen (PSA) screening and the morbidity of prostate biopsies, several blood- and urine-based biomarkers have been proposed for pre-biopsy risk stratification. These assays aim to reduce the frequency of unnecessary biopsies (i.e., negative or Grade Group 1 [GG1]) while maintaining highly sensitive detection of clinically significant cancer (GG ≥ 2) prostate cancer. METHODS: We reviewed the literature describing the use of currently available blood- and urine-based biomarkers for detection of GG ≥ 2 cancer, including the Prostate Health Index (PHI), 4Kscore, MyProstateScore (MPS), SelectMDx, ExoDx Prostate Intelliscore (EPI), and IsoPSA. To facilitate clinical application, we focused on the use of biomarkers as a post-PSA secondary test prior to biopsy, as proposed in clinical guidelines. Our outcomes included test performance measures-sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV)-as well as clinical outcomes resulting from biomarker use (i.e., unnecessary biopsies avoided, GG ≥ 2 cancers missed). RESULTS: Contemporary validation data (2015-2023) reveal that currently available biomarkers provide ~15-50% specificity at a sensitivity of 90-95% for GG ≥ 2 PCa. Clinically, this indicates that secondary use of biomarker testing in men with elevated PSA could allow for avoidance of up to 15-50% of unnecessary prostate biopsies, while preserving detection of 90-95% of GG ≥ 2 cancers that would be detected under the traditional "biopsy all" approach. CONCLUSIONS: The contemporary literature further supports the proposed role of post-PSA biomarker testing to reduce the use of invasive biopsy while maintaining highly sensitive detection of GG ≥ 2 cancer. Questions remain regarding the optimal application of biomarkers in combination or in sequence with mpMRI.

Electrochemical Impedance-Based Biosensors for the Label-Free Detection of the Nucleocapsid Protein from SARS-CoV-2.

Diagnosis of coronavirus disease (COVID-19) is important because of the emergence and global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time polymerase chain reaction (PCR) is widely used to diagnose COVID-19, but it is time-consuming and requires sending samples to test centers. Thus, the need to detect antigens for rapid on-site diagnosis rather than PCR is increasing. We quantified the nucleocapsid (N) protein in SARS-CoV-2 using an electro-immunosorbent assay (El-ISA) and a multichannel impedance analyzer with a 96-interdigitated microelectrode sensor (ToAD). The El-ISA measures impedance signals from residual detection antibodies after sandwich assays and thus offers highly specific, label-free detection of the N protein with low cross-reactivity. The ToAD sensor enables the real-time electrochemical detection of multiple samples in conventional 96-well plates. The limit of detection for the N protein was 0.1 ng/mL with a detection range up to 10 ng/mL. This system did not detect signals for the S protein. While this study focused on detecting the N protein in SARS-CoV-2, our system can also be widely applicable to detecting various biomolecules involved in antigen-antibody interactions.

Nanomaterial-based Optical and Electrochemical Biosensors for Amyloid beta and Tau: Potential for early diagnosis of Alzheimer's Disease.

INTRODUCTION: Alzheimer's disease (AD), a heterogeneous pathological process representing the most common causes of dementia worldwide, has required early and accurate diagnostic tools. Neuropathological hallmarks of AD involve the aberrant accumulation of Amyloid beta (Aß) into Amyloid plaques and hyperphosphorylated Tau into neurofibrillary tangles, occurring long before the onset of brain dysfunction.Areas covered:Considering the significance of Aß and Tau in AD pathogenesis, these proteins have been adopted as core biomarkers of AD, and their quantification has provided precise diagnostic information to develop next-generation AD therapeutic approaches. However, conventional diagnostic methods may not suffice to achieve clinical criteria that are acceptable for proper diagnosis and treatment. The advantages of nanomaterial-based biosensors including facile miniaturization, mass fabrication, ultra-sensitivity, make them useful to be promising tools to measure Aß and Tau simultaneously for accurate validation of low-abundance yet potentially informative biomarkers of AD.. EXPERT OPINION: The study has identified the potential application of advanced biosensors as standardized clinical diagnostic tools for AD, evolving the way for new and efficient AD control with minimum economic and social burden. After clinical trial, nanobiosensors for measuring Aß and Tau simultaneously possess innovative diagnosis of AD to provide significant contributions to primary Alzheimer's care intervention.

Nanobiosensors for Non-Amyloidbeta-Tau Biomarkers as Advanced Reporters of Alzheimer's Disease.

Emerging nanomaterials providing benefits in sensitivity, specificity and cost-effectiveness are being widely investigated for biosensors in the application of Alzheimer's disease (AD) diagnosis. Core biomarkers amyloid-beta (Aß) and Tau have been considered as key neuropathological hallmarks of AD. However, they did not sufficiently reflect clinical severity and therapeutic response, proving the difficulty of the Aß- and Tau-targeting therapies in clinical trials. In recent years, there has still been a shortage of sensors for non-Aß-Tau pathophysiological biomarkers that serve as advanced reporters for the early diagnosis of AD, predict AD progression, and monitor the treatment response. Nanomaterial-based sensors measuring multiple non-Aß-Tau biomarkers could improve the capacity of AD progression characterization and supervised treatment, facilitating the comprehensive management of AD. This is the first review to principally represent current nanobiosensors for non-Aß-Tau biomarker and that strategically deliberates future perspectives on the merit of non-Aß-Tau biomarkers, in combination with Aß and Tau, for the accurate diagnosis and prognosis of AD.

Robust Magnetized Graphene Oxide Platform for In Situ Peptide Synthesis and FRET-Based Protease Detection.

Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and ß-secretase in a concentration-dependent manner. Particularly, ß-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.

Microfluidic Generation of Amino-Functionalized Hydrogel Microbeads Capable of On-Bead Bioassay.

Microfluidic generation of hydrogel microbeads is a highly efficient and reproducible approach to create various functional hydrogel beads. Here, we report a method to prepare crosslinked amino-functionalized polyethylene glycol (PEG) microbeads using a microfluidic channel. The microbeads generated from a microfluidic device were evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and confocal laser scanning microscopy, respectively. We found that the microbeads were monodisperse and the amino groups were localized on the shell region of the microbeads. A swelling test exhibited compatibility with various solvents. A cell binding assay was successfully performed with RGD peptide-coupled amino-functionalized hydrogel microbeads. This strategy will enable the large production of the various functional microbeads, which can be used for solid phase peptide synthesis and on-bead bioassays.

Amyloid beta in nasal secretions may be a potential biomarker of Alzheimer's disease.

We investigated the level of amyloid beta (Aß) in nasal secretions of patients with Alzheimer's disease dementia (ADD) using interdigitated microelectrode (IME) biosensors and determined the predictive value of Aß in nasal secretions for ADD diagnosis. Nasal secretions were obtained from 35 patients with ADD, 18 with cognitive decline associated with other neurological disorders (OND), and 26 cognitively unimpaired (CU) participants. Capacitance changes in IMEs were measured by capturing total Aß (ΔCtAß). After 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) was injected, additional capacitance changes due to the smaller molecular weight Aß oligomers disassembled from the higher molecular weight oligomeric Aß were determined (ΔCoAß). By dividing two values, the capacitance ratio (ΔCoAß/ΔCtAß) was determined and then normalized to the capacitance change index (CCI). The CCI was higher in the ADD group than in the OND (p = 0.040) and CU groups (p = 0.007). The accuracy of the CCI was fair in separating into the ADD and CU groups (area under the receiver operating characteristic curve = 0.718, 95% confidence interval = 0.591-0.845). These results demonstrate that the level of Aß in nasal secretions increases in ADD and the detection of Aß in nasal secretions using IME biosensors may be possible in predicting ADD.

Electrochemical Detection of C-Reactive Protein in Human Serum Based on Self-Assembled Monolayer-Modified Interdigitated Wave-Shaped Electrode.

An electrochemical capacitance immunosensor based on an interdigitated wave-shaped micro electrode array (IDWµE) for direct and label-free detection of C-reactive protein (CRP) was reported. A self-assembled monolayer (SAM) of dithiobis (succinimidyl propionate) (DTSP) was used to modify the electrode array for antibody immobilization. The SAM functionalized electrode array was characterized morphologically by atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). The nature of gold-sulfur interactions on SAM-treated electrode array was probed by X-ray photoelectron spectroscopy (XPS). The covalent linking of anti-CRP-antibodies onto the SAM modified electrode array was characterized morphologically through AFM, and electrochemically through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The application of phosphate-buffered saline (PBS) and human serum (HS) samples containing different concentrations of CRP in the electrode array caused changes in the electrode interfacial capacitance upon CRP binding. CRP concentrations in PBS and HS were determined quantitatively by measuring the change in capacitance (ΔC) through EIS. The electrode immobilized with anti-CRP-antibodies showed an increase in ΔC with the addition of CRP concentrations over a range of 0.01-10,000 ng mL-1. The electrode showed detection limits of 0.025 ng mL-1 and 0.23 ng mL-1 (S/N = 3) in PBS and HS, respectively. The biosensor showed a good reproducibility (relative standard deviation (RSD), 1.70%), repeatability (RSD, 1.95%), and adequate selectivity in presence of interferents towards CRP detection. The sensor also exhibited a significant storage stability of 2 weeks at 4 °C in 1× PBS.

Gold Nanoparticle-Stabilized, Tyrosine-Rich Peptide Self-Assemblies and Their Catalytic Activities in the Reduction of 4-Nitrophenol.

Peptides are suitable candidates for templates in the fabrication of various metal nanoparticles (NPs) because of their metal-binding ability and templating effect, which impart physicochemical properties to the produced nanoparticles. Peptide-binding gold nanoparticles (AuNPs) show high catalytic activity that permits their application in oxidation or reduction reactions. Herein, we prepared morphology-controllable AuNPs stabilized by self-assembled tyrosine-rich peptides (YC7) by varying the pH and YC7 peptide/Au3+ concentration ratio in 2-( N-morpholino)ethanesulfonic acid (MES) buffer solution. The catalytic activities of the YC7 peptide-stabilized AuNPs (YC7@AuNPs) were tested for 4-nitrophenol (4-NP) reduction, and kinetic analysis was performed to calculate the apparent rate constants and activation energies. The relatively low activation energy of the YC7@AuNPs could be explained by the hypothesis that the tyrosine-moiety of YC7 enriches the electron density of Au metal.

An Enhanced Platform to Analyse Low-Affinity Amyloid ß Protein by Integration of Electrical Detection and Preconcentrator.

Sensitivity and limit of detection (LOD) enhancement are essential criteria for the development of ultrasensitive molecular sensors. Although various sensor types have been investigated to enhance sensitivity and LOD, analyte detection and its quantification are still challenging, particularly for protein-protein interactions with low association constants. To solve this problem, here, we used ion concentration polarization (ICP)-based preconcentration to increase the local concentration of analytes in a microfluidic platform for LOD improvement. This was the first demonstration of a microfluidic device with an integrated ICP preconcentrator and interdigitated microelectrode (IME) sensor to detect small changes in surface binding between antigens and antibodies. We detected the amyloid beta (Aß) protein, an Alzheimer's disease marker, with low binding affinity to its antibodies by adopting ICP preconcentration phenomena. We demonstrated that a combination of ICP preconcentrator and IME sensor increased the LOD by 13.8-fold to femtomolar level (8.15 fM), which corresponds to a significant advance for clinical applications.

A highly sensitive plasma-based amyloid-ß detection system through medium-changing and noise cancellation system for early diagnosis of the Alzheimer's disease.

We developed an interdigitated microelectrode (IME) sensor system for blood-based Alzheimer's disease (AD) diagnosis based on impedimetric detection of amyloid-ß (Aß) protein, which is a representative candidate biomarker for AD. The IME sensing device was fabricated using a surface micromachining process. For highly sensitive detection of several tens to hundreds of picogram/mL of Aß in blood, medium change from plasma to PBS buffer was utilized with signal cancellation and amplification processing (SCAP) system. The system demonstrated approximately 100-folds higher sensitivity according to the concentrations. A robust antibody-immobilization process was used for stability during medium change. Selectivity of the reaction due to the affinity of Aß to the antibody and the sensitivity according to the concentration of Aß were also demonstrated. Considering these basic characteristics of the IME sensor system, the medium change was optimized in relation to the absolute value of impedance change and differentiated impedance changes for real plasma based Aß detection. Finally, the detection of Aß levels in transgenic and wild-type mouse plasma samples was accomplished with the designed sensor system and the medium-changing method. The results confirmed the potential of this system to discriminate between patients and healthy controls, which would enable blood-based AD diagnosis.

Fabrication of Localized Surface Plasmon Resonance Sensor Based on Optical Fiber and Micro Fluidic Channel.

This paper proposes Fiber-Optic Localized Surface Plasmon Resonance (FO LSPR) sensor combined with a micro fluidic channel, which enables continuous supply of fluid for bio-reaction. The proposed method prevents degradation of the sensing performance due to changes in measurement conditions. The feasibility of the FO LSPR sensor with a micro fluidic channel was demonstrated by computational fluid dynamics (CFD) simulation. Also, the proposed method was assessed by measuring the output intensity of the FO LSPR sensor at various refractive index solutions. Finally, a prostate-specific antigen (PSA) immunoassay was performed to evaluate the possibility of the fabricated sensor system as a biosensor.

Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvß3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

A highly sensitive, direct and label-free technique for Hg(2+) detection using Kelvin probe force microscopy.

For several decades, various nanomaterials have been used in a wide range of industrial fields, research areas, and commercial products. Among many nanomaterials, nano-sized mercury materials are one of the most widely used nanomaterials in real life. However, due to the high toxicity of Hg(2+), it is imperative to develop an effective and practical detection method for Hg(2+) to protect human health and environment. In this study, a highly sensitive, label-free method of detecting Hg(2+) that requires only a single drop of solution was developed. The detection mechanism is based on the different surface potential arising from Hg(2+) binding to mismatched thymine-thymine sequences, creating a very stable base pair. The surface potential is measured with Kelvin probe force microscopy (KPFM) to a molecular resolution. The developed method is capable of detecting 2 fmol of Hg(2+), which is 500 times more sensitive than previously reported techniques. Moreover, our method can selectively detect Hg(2+) and can also be applied to tap water and river water. This KPFM-based Hg(2+) detection method can be used as an early detection technique for practical applications.

Label-free detection of zinc oxide nanowire using a graphene wrapping method.

Zinc oxide nanowires (ZnO NWs) have been attempted to various applications, such as piezoelectric devices, energy harvesting devices, self-powered nanosensors, and biomedical devices. However, recent reports have shown the toxic effect of ZnO NWs. In this report, we described the detection of ZnO NWs, for the first time using reduced graphene oxide (RGO) wrapping method. By wrapping RGO to ZnO NW (RGO-ZnO NW), we are able to aggregate ZnO NWs and increase the sensing performance. The detection measurement is based on the resonance frequency shift derived from mass variation of RGO-ZnO NW adsorption on the DNA immobilized resonator. The resonator is able to detect ZnO NWs with detection limit of 100 ng mL(-1) which is 2 order below the fatal toxic concentration of ZnO NWs in Human Monocyte Macrophages (HMMs). Furthermore, the resonator is able to detect ZnO NWs in real tap water, showing the potential as ZnO NWs screening platform in real environmental aqua system.

Nanomechanical measurement of astrocyte stiffness correlated with cytoskeletal maturation.

Astrocytes are known to serve as scaffolding cells that shape the brain. The physical properties of astrocytes, such as stiffness, are important for their scaffolding function. These properties may be altered in certain pathological conditions, such as in brain cancer. However, actual stiffness of astrocytes is not yet well understood. Here, we report that the astrocyte stiffness is positively correlated with the density of cytoskeletal proteins, such as actin filaments, microtubules, and intermediate filaments. The value of the stiffness of astrocytes as measured by atomic force microscopy (AFM) increases 38-fold in five-week-old rats compared to postnatal-day zero pups. Using multicolor confocal microscopy, we found that the complexity of cytoskeletal proteins, such as actin filaments, microtubules, and intermediate filaments, increase as the animal gets older. Our findings indicate that the change of stiffness positively correlates with the maturation of cytoskeletal proteins, and suggest that AFM can be useful as an analytical and diagnostic tool for neuroscience.

A micro-fabricated force sensor using an all thin film piezoelectric active sensor.

The ability to measure pressure and force is essential in biomedical applications such as minimally invasive surgery (MIS) and palpation for detecting cancer cysts. Here, we report a force sensor for measuring a shear and normal force by combining an arrayed piezoelectric sensors layer with a precut glass top plate connected by four stress concentrating legs. We designed and fabricated a thin film piezoelectric force sensor and proposed an enhanced sensing tool to be used for analyzing gentle touches without the external voltage source used in FET sensors. Both the linear sensor response from 3 kPa to 30 kPa and the exact signal responses from the moving direction illustrate the strong feasibility of the described thin film miniaturized piezoelectric force sensor.

Synthesis of silver and gold nanoparticles using cashew nut shell liquid and its antibacterial activity against fish pathogens.

This study reveals a green process for the production of multi-morphological silver (Ag NPs) and gold (Au NPs) nanoparticles, synthesized using an agro-industrial residue cashew nut shell liquid. Aqueous solutions of Ag(+) ions for silver and chloroaurate ions for gold were treated with cashew nut shell extract for the formation of Ag and Au NPs. The nano metallic dispersions were characterized by measuring the surface plasmon absorbance at 440 and 546 nm for Ag and Au NPs. Transmission electron microscopy showed the formation of nanoparticles in the range of 5-20 nm for silver and gold with assorted morphologies such as round, triangular, spherical and irregular. Scanning electron microscopy with energy dispersive spectroscopy and X-ray diffraction analyses of the freeze-dried powder confirmed the formation of metallic Ag and Au NPs in crystalline form. Further analysis by Fourier transform infrared spectroscopy provided evidence for the presence of various biomolecules, which might be responsible for the reduction of silver and gold ions. The obtained Ag and Au NPs had significant antibacterial activity, minimum inhibitory concentration and minimum bactericidal concentration on bacteria associated with fish diseases.

Ultra-sensitive direct detection of silver ions via Kelvin probe force microscopy.

Nanotoxicity is receiving great importance due to its potential impact on human health and environment and due to rapid development in the field of nanoscale research and industry. Herein, we report the Kelvin probe force microscope (KPPM)-based nanotoxicity material detection using surface potential difference. In general, it is difficult to measure the size of ion (Ag(+)) using a conventional atomic force microscope (AFM) because of the limited resolution. In this study, we have demonstrated that KPFM is capable of ultra-sensitive detection of silver ion with silver specific DNA by a single droplet. Furthermore, the measured surface potentials for Ag+ and DNA binding enable the detection performance for a practical sample that is general drinking water. Remarkably, the KPFM based silver ion detection enables an insight into the coordination chemistry, which plays an important role in early detection of toxicity. This implies that KPFM based detection system opens a new avenue for water testing sensor.