Design and Application of Metal Organic Framework ZIF-90-ZnO-MoS2 Nanohybrid for an Integrated Electrochemical Liquid Biopsy.

Limited healthcare capacity highlights the needs of integrated sensing systems for personalized health-monitoring. However, only limited sensors can be employed for point-of-care applications, emphasizing the lack of a generalizable sensing platform. Here, we report a metal organic framework (MOF) ZIF-90-ZnO-MoS2 nanohybrid-based integrated electrochemical liquid biopsy (ELB) platform capable of direct profiling cancer exosomes from blood. Using a bottom-up approach for sensor design, a series of critical sensing functions is considered and encoded into the MOF material interface by programming the material with different chemical and structural features. The MOF-based ELB platform is able to achieve one-step sensor fabrication, target isolation, nonfouling and high-sensitivity sensing, direct signal transduction, and multiplexed detection. We demonstrated the capability of the designed sensing system on differentiating cancerous groups from healthy controls by analyzing clinical samples from lung cancer patients, providing a generalizable sensing platform.

Phase-Regulated Sensing Mechanism of MoS2 Based Nanohybrids toward Point-of-Care Prostate Cancer Diagnosis.

Alpha-methylacyl-CoA racemase (AMACR) has been proven to be consistently overexpressed in prostate cancer epitheliums, and is expected to act as a positive biomarker for the diagnosis of prostate carcinoma in clinical practice. Here, a strategy for specific determination of AMACR in real human serum by using an electrochemical microsensor system is presented. In order to implement the protocol, a self-organized nanohybrid consisting of metal nanopillars in a 2D MoS2 matrix is developed as material for the sensing interface. The testing signal outputs are strongly enhanced with the presence of the nanohybrids owing to that the metal pillars provide an efficient mass difussion and electron transfer path to the MoS2 film surface. Furthermore, the phase-regulated sensing mechanism over MoS2 is noticed and demonstrated by density functional theory calculation and experiments. The explored MoS2 based nanohybrids are employed for the fabrication of an electrochemical microsensor, presenting good linear relationship in both ng µL-1 and pg µL-1 ranges for AMACR quantification. The sampling analysis of human serum indicates that this microsensor has good diagnostic specificity and sensitivity toward AMACR. The proposed electrochemical microsensor system also demonstrates the advantages of convenience, cost-effectiveness, and disposability, resulting in a potential integrated microsystem for point-of-care prostate cancer diagnosis.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Dynamic Control of Peptide Strand Displacement Reaction Using Functional Biomolecular Domain for Biosensing.

Nature's great repository provides nucleic acids and amino acids as the fundamental elements of life. Inspired by the programmability of nucleic acids, DNA nanotechnology has been extensively developed based on the strand displacement reaction of nucleic acids. In comparison with nucleic acids, amino acids possess higher programmability and more functionalities owing to the diversity of the amino acid unit. However, the design of the peptide-based bimolecular cascade is still limited. We herein describe a peptide-based strand displacement reaction, which was granted with a specific biological function by addition of a functional domain onto the coiled-coil peptide based displacement substrate. The displacement substrate was specifically designed to response to Tau protein based on a well-established Tau inhibition sequence. We demonstrated that the kinetics of the designed displacement reaction can be dynamically tuned through blocking the toehold region to prevent migration. A nanomolar Tau detection linear range was achieved through the designed displacement reaction within a rapid turnaround time of 30 min. We also presented the capability of the peptide strand displacement based sensing system operating in real human biological samples and its excellent orthogonality on response to irrelevant biological components. We envision that this will be of especially high utility for the development of next-generation biotechnology.

Neutral Charged Immunosensor Platform for Protein-based Biomarker Analysis with Enhanced Sensitivity.

A noninvasive, highly sensitive universal immunosensor platform for protein-based biomarker detection is described in this Article. A neutral charged sensing environment is constructed by an antibody with an oppositely charged amino acid as surface charge neutralizer. By adjusting the pH condition of the testing environment, this neutral charged immunosensor (NCI) directly utilizes the electrostatic charges of the analyte for quantification of circulating protein markers, achieving a wide dynamic range covering through the whole picomole level. Comparing with previous studies on electrostatic charges characterization, this NCI demonstrates its capability to analyze not only the negatively charged biomolecules but also positively charged analytes. We applied this NCI for the detection of HE4 antigen with a detection limit at 2.5 pM and Tau antigen with a detection limit at 0.968 pM, demonstrating the high-sensitivity property of this platform. Furthermore, this NCI possesses a simple fabrication method (less than 2 h) and a short testing turnaround time (less than 30 min), providing an excellent potential for further clinical point-of-care applications.

Advanced fabrication of biosensor on detection of Glypican-1 using S-Acetylmercaptosuccinic anhydride (SAMSA) modification of antibody.

Glypican-1 (GPC-1) has been recognized as biomarker of pancreatic cancer. Quantification of GPC-1 level is also pivotal to breast cancer and prostate cancer's patients. We hereby report the first biosensor for GPC-1 detection. Instead of using crosslinking technique and surface immobilization of antibody, we applied a novel method for biosensor fabrication, using S-Acetylmercaptosuccinic anhydride (SAMSA) to modify the Anti-GPC-1 producing a thiol-linked Anti-GPC-1. The thiol-linked Anti-GPC-1 was then directly formed a single-layer antibody layer on the gold biosensor, minimizing the biosensor preparation steps significantly. Time of Flight Secondary Ions Mass Spectroscopy (TOF-SIMS) characterization verified the thiol-linked antibody layer and demonstrated a unique perspective for surface protein characterization. Differential pulse voltammetry (DPV) was applied to quantify GPC-1 antigen in undiluted human serum with a concentration range of 5,000 pg/µL to 100 pg/µL. The performance of this newly designed biosensor was also compared with modified self-assembled monolayer system fabricated biosensor, demonstrating the high-sensitivity and high-reproducibility of the SAMSA modified antibody based biosensor. This simple fabrication method can also expand to detection of other biomolecules. The simplified operation process shows great potential in clinical application development.

Bioconjugated, Single-Use Biosensor for the Detection of Biomarkers of Prostate Cancer.

Prostate cancer is prevalent among cancers in men. A simple method for screening of reliable biomarkers is pivotal for early detection of prostate cancer.  Prostate-specific antigen (PSA) has been a commonly used biomarker for prostate cancer, in spite of its false-positive limitation. On the other hand, alpha-methylacyl-CoA racemase (AMACR), a metabolic enzyme, has been proven to be a highly expressed biomarker in prostate cancer cells. Therefore, a method or tool, which can detect either PSA or AMACR or both simply, cost effectively, and with high sensitivity and selectivity is desirable. We describe a novel bioconjugated, single-use biosensor capable of detecting both PSA and AMACR antigens in undiluted human serum. The preparation of the biosensor by the bioconjugation mechanism occurred within a day, which could be completed prior to actual testing. The effectiveness of the bioconjugation mechanism and the coverage of the electrode surface of the biosensor were experimentally assessed. Measurements of PSA and AMACR antigens and the specificity of the biosensor were carried out using differential pulse voltammetry. This biosensor was single-use and cost-effective and required a small quantity of test medium and relatively short preparation time, providing a very attractive biosensor for the detection of the biomarkers of prostate cancer.

Detection of 17 ß-Estradiol in Environmental Samples and for Health Care Using a Single-Use, Cost-Effective Biosensor Based on Differential Pulse Voltammetry (DPV).

Environmental estrogen pollution and estrogen effects on the female reproductive system are well recognized scientifically. Among the estrogens, 17 ß-estradiol is a priority in environmental estrogen pollution, and it is also a major contributor to estrogen which regulates the female reproductive system. 17 ß-estradiol is carcinogenic and has a tumor promotion effect relating to breast cancer, lung cancer and others. It also affects psychological well-being such as depression, fatigue and others. Thus, a simple method of detecting 17 ß-estradiol will be important for both environmental estrogen pollution and health care. This study demonstrates a single-use, cost-effective 17 ß-estradiol biosensor system which can be used for both environmental and health care applications. The bio-recognition mechanism is based on the influence of the redox couple, K3Fe(CN)6/K4Fe(CN)6 by the interaction between 17 ß-estradiol antigen and its α-receptor (ER-α; α-estrogen antibody). The transduction mechanism is an electrochemical analytical technique, differential pulse voltammetry (DPV). The levels of 17 ß-estradiol antigen studied were between 2.25 pg/mL and 2250 pg/mL; Phosphate buffered saline (PBS), tap water from the Cleveland regional water district, and simulated urine were used as the test media covering the potential application areas for 17 ß-estradiol detection. An interference study by testosterone, which has a similar chemical structure and molecular weight as those of 17 ß-estradiol, was carried out, and this 17 ß-estradiol biosensor showed excellent specificity without any interference by similar chemicals.

Bimetallic PtM (M=Pd, Ir) nanoparticle decorated multi-walled carbon nanotube enzyme-free, mediator-less amperometric sensor for H2O2.

A new highly catalytic and intensely sensitive amperometric sensor based on PtM (where M=Pd, Ir) bimetallic nanoparticles (NPs) for the rapid and accurate estimation of hydrogen peroxide (H(2)O(2)) by electrooxidation in physiological conditions is reported. PtPd and PtIr NPs-decorated multiwalled carbon nanotube nanocatalysts (PtM/MWCNTs) were prepared by a modified Watanabe method, and were characterized by XRD, TEM, ICP, and XAS. The sensors were constructed by immobilizing PtM/MWCNTs nanocatalysts in a Nafion film on a glassy carbon electrode. Both PtPd/MWCNTs and PtIr/MWCNTs assemblies catalyzed the electrochemical oxidation of H(2)O(2). Cyclic voltammetry characterization measurements revealed that both the PtM (M=Pd, Ir)/MWCNTs/GCE possessed similar electrochemical surface areas (∼0.55 cm(2)), and electron transfer rate constants (∼1.23 × 10(-3)cms(-1)); however, the PtPd sensor showed a better performance in H(2)O(2) sensing than did the PtIr counterpart. Explanations were sought from XAS measurements to explain the reasons for differences in sensor activity. When applied to the electrochemical detection of H(2)O(2), the PtPd/MWCNTs/GC electrode exhibited a low detection limit of 1.2 µM with a wide linear range of 2.5-125 µM (R(2)=0.9996). A low working potential (0V (SCE)), fast amperometric response (<5s), and high sensitivity (414.8 µA mM(-1)cm(-2)) were achieved at the PtPd/MWCNTs/GC electrode. In addition, the PtPd/MWCNTs nanocatalyst sensor electrode also exhibited excellent reproducibility and stability. Along with these attractive features, the sensor electrode also displayed very high specificity to H(2)O(2) with complete elimination of interference from UA, AA, AAP and glucose.

Detection of Alpha-Methylacyl-CoA Racemase (AMACR), a Biomarker of Prostate Cancer, in Patient Blood Samples Using a Nanoparticle Electrochemical Biosensor.

Although still commonly used in clinical practice to screen and diagnose prostate cancer, there are numerous weaknesses of prostate-specific antigen (PSA) testing, including lack of specificity and the inability to distinguish between aggressive and indolent cancers. A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has been previously demonstrated to distinguish cancer from healthy and benign prostate cells with high sensitivity and specificity. However, no accurate clinically useful assay has been developed. This study reports the development of a single use, disposable biosensor for AMACR detection. Human blood samples were used to verify its validity, reproducibility and reliability. Plasma samples from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia (HGPIN), and 5 prostate cancer patients were measured for AMACR levels. The average AMACR levels in the prostate cancer patients was 10 fold higher (mean(SD) = 0.077 (0.10)) than either the controls (mean(SD) = 0.005 (0.001)) or HGPIN patients (mean(SD) = 0.004 (0.0005)). At a cutoff of between 0.08 and 0.9, we are able to achieve 100% accuracy in separating prostate cancer patients from controls. Our results provide strong evidence demonstrating that this biosensor can perform as a reliable assay for prostate cancer detection and diagnosis.

A thick-film sensor as a novel device for determination of polyphenols and their antioxidant capacity in white wine.

A thick-film electrochemical sensor with an iridium-carbon working electrode was used for determining polyphenols and their antioxidant capacity in white wine. Caffeic acid was used as a model species because it has the ability to produce the highest oxidation current. The correlation coefficient of 0.9975 was obtained between sensor response and caffeic acid content. The total phenolic content (TPC) and scavenging activity on 1,1-diphenyl-2-pycrylhydrazyl (DPPH·) radical were also found to be strongly correlated with the concentration of caffeic acid, with a correlation coefficient of 0.9823 and 0.9958, respectively. The sensor prototype was proven to be a simple, efficient and cost effective device to evaluate the antioxidant capacity of substances.

Development of a dimethyl ether (DME) sensor using platinum nanoparticles and thick-film printing.

A portable and cost-effective technique to measure the dimethyl ether (DME) concentrations has been developed. It is based on an electrochemical principle measuring the oxidation current of DME at an applied potential of +0.2V versus a Ag/AgCl reference electrode. Thick-film printing technique is used for the fabrication of this DME sensor, and platinum nanoparticles in the crystallite size of 5.5 nm are used for the modification of the working electrode surface. This modification enhances the sensor performance significantly leading to a higher sensitivity of the sensor comparing to bare platinum electrode. Evaluation and characterization of this sensor are carried out over the DME concentration range of 0-7% (v/v), and a linear relationship between sensor outputs and the DME concentrations with an average R(2) of 0.996 exists. The reproducibility of the sensor is also very good. This electrochemically based DME sensor fabricated by thick-film screen printing technique and using the platinum nanoparticles to enhance its performance will be valuable and practical for the estimation of the airway mucosal blood flow.

Estrogen acidifies vaginal pH by up-regulation of proton secretion via the apical membrane of vaginal-ectocervical epithelial cells.

The objective of this study was to assess estrogen-dependent cellular mechanisms that could contribute to the acid pH of the vaginal lumen. Cultures of normal human cervical-vaginal epithelial (hECE) cells and endocervical cells were grown on filters, and acidification of the extracellular solutions on the luminal (L-pHo) and contraluminal (CL-pHo) sides was measured. The hECE cells and endocervical cells decreased CL-pHo from 7.40 to 7.25 within 20-30 min of incubation in basic salt solution. Endocervical cells also produced a similar decrease in L-pHo. In contrast, hECE cells acidified L-pHo down to pH 7.05 when grown as monoculture and down to pH 6.05 when grown in coculture with human cervical fibroblasts. This enhanced acid secretion into the luminal compartment was estrogen dependent because removal of endogenous steroid hormones attenuated the effect, whereas treatment with 17beta-estradiol restored it. The 17beta-estradiol effect was dose dependent (EC50 0.5 nm) and could be mimicked by diethylstilbestrol and in part by estrone and tamoxifen. Preincubation with ICI-182780, but not with progesterone, blocked the estrogen effect. Preincubation of cells with the V-ATPase blocker bafilomycin A1, when administered to the luminal solution, attenuated the baseline and estrogen-dependent acid secretion into the luminal solution. Treatment with EGTA, to abrogate the tight junctional resistance, blocked the decrease in L-pHo and stimulated a decrease in CL-pHo, indicating that the tight junctions are necessary for maintaining luminal acidification. We conclude that vaginal-ectocervical cells acidify the luminal canal by a mechanism of active proton secretion, driven in part by V-H+-ATPase located in the apical plasma membrane and that the baseline active net proton secretion occurs constitutively throughout life and that this acidification is up-regulated by estrogen.

Amperometric acetylcholine sensor catalyzed by nickel anode electrode.

An amperometric method was using a nickel catalytic electrode in aqueous base solution for detecting acetylcholine (ACh). A sensing mechanism was developed in which ACh was hydrolyzed in base aqueous solution to produce the acetic anion and choline. The alcohol group of choline was oxidized to the corresponding carboxylic acid by Ni(OH)2/NiOOH catalytic system. The amperometric response resulted from the current generated by ACh oxidation in response to step changes in ACh concentration. The potential window of limiting current of ACh anodic oxidation at the Ni interface was determined in NaOH electrolyte. The effect of NaOH electrolyte concentration on sensitivity was also discussed. At the optimum operating condition, the method exhibits a good linear relationship between the response current and the ACh concentration. The response time of the ACh sensing system was 10 s. Scanning electrochemical microscopy (SECM) with platinum micro-tips was used to investigate the diffusion layer thickness of Ni electrode.