Jian Shu

Bioresponsive Release System for Visual Fluorescence Detection of Carcinoembryonic Antigen from Mesoporous Silica Nanocontainers Mediated Optical Color on Quantum Dot-Enzyme-Impregnated Paper

An all-in-one paper-based analytical device (PAD) was successfully developed for visual fluorescence detection of carcinoembryonic antigen (CEA) on CdTe/CdSe quantum dot (QD)-enzyme-impregnated paper by coupling with a bioresponsive controlled-release system from DNA-gated mesoporous silica nanocontainers (MSNs). The assay was carried out in a centrifuge tube by using glucose-loaded MSNs with a CEA aptamer and a QD-enzyme-paper attached on the lid. Initially, single-strand complementary DNA to a CEA aptamer was covalently conjugated to the aminated MSN, and then glucose (enzyme substrate) molecules were gated into the pore with the help of the aptamer. Glucose oxidase (GOD) and CdTe/CdSe QDs were coimmobilized on paper for the visual fluorescence signal output. Upon target CEA introduction in the detection cell, the analyte specifically reacted with the immobilized aptamer on the MSN to open the pore, thereby resulting in the glucose release. The released glucose was oxidized by the immobilized GOD on paper to produce gluconic acid and hydrogen peroxide, and the latter quenched the fluorescence of CdTe/CdSe QDs, which could be determined by the naked eye on a portable smartphone and a commercial fluorospectrometer. Under optimal conditions, the PAD-based sensing system enabled sensitive discrimination of target CEA against other biomarkers or proteins in a linear range of 0.05-20 ng mL-1 with a limit of detection of 6.7 pg mL-1 (ppt). In addition, our strategy displayed high specificity, good reproducibility, and acceptable accuracy for analyzing human serum specimens with a commercial human CEA ELISA kit. Importantly, this methodology offers promise for simple analysis of biological samples and is suitable for use in the mass production of miniaturized devices, thus opening new opportunities for protein diagnostics and biosecurity.

Near-Infrared-to-Ultraviolet Light-Mediated Photoelectrochemical Aptasensing Platform for Cancer Biomarker Based on Core-Shell NaYF4:Yb,Tm@TiO2 Upconversion Microrods

Titanium dioxide (TiO2; as a potential photosensitizer) has good photocurrent performance and chemical stability but often exhibits low utilization efficiency under ultraviolet (UV) region excitation. Herein, we devised a near-infrared light-to-UV light-mediated photoelectrochemical (PEC) aptasensing platform for the sensitive detection of carcinoembryonic antigen (CEA) based on core-shell NaYF4:Yb,Tm@TiO2 upconversion microrods by coupling with target-triggered rolling circle amplification (RCA). The upconversion microrods synthesized through the hydrothermal reaction could act as a photosensing platform to convert the near-infrared (near-IR) excitation into UV emission for generation of photoinduced electrons. The target analyte was determined on a functional magnetic bead by using the corresponding aptamers with a sandwich-type assay format. Upon target CEA introduction, a complex was first formed between capture aptamer-1-conjugated magnetic bead (Apt1-MB) and aptamer-2-primer DNA (Apt2-pDNA). Thereafter, the carried primer DNA by the aptamer-2 paired with linear padlock DNA to trigger the RCA reaction. The guanine (G)-rich product by RCA reaction was cleaved by exonuclease I and exonuclease III (Exos I/III), thereby resulting in the formation of numerous individual guanine bases to enhance the photocurrent of core-shell NaYF4:Yb,Tm@TiO2 upconversion microrods under near-IR illumination (980 nm). Under optimal conditions, the near-IR light-mediated PEC aptasensing system could exhibit good photoelectrochemical response toward target CEA and allowed for the detection of target CEA as low as 3.6 pg mL-1. High reproducibility and good accuracy were achieved for analysis of human serum specimens. Importantly, the near-IR-activated PEC aptasensing scheme provides a promising platform for ultrasensitive detection of other biomolecules.

Enzymatic Oxydate-Triggered Self-Illuminated Photoelectrochemical Sensing Platform for Portable Immunoassay Using Digital Multimeter

Herein a novel split-type photoelectrochemical (PEC) immunosensing platform was designed for sensitive detection of low-abundance biomarkers (prostate-specific antigen, PSA, used in this case) by coupling a peroxyoxalate chemiluminescence (PO-CL) self-illuminated system with digital multimeter (DMM) readout. The PEC detection device consisted of a capacitor/DMM-joined electronic circuit and a PO-CL-based self-illuminated cell. Initially, reduced graphene oxide-doped BiVO4 (BiVO4-rGO) photovoltaic materials with good photoelectric properties was integrated into the capacitor/DMM-joined circuit for photocurrent generation in the presence of hydrogen peroxide (H2O2, as the hole-trapping reagent). A sandwich-type immunoreaction with target PSA was carried out in capture antibody-coated microplates by using glucose oxidase/detection antibody-conjugating gold nanoparticle (pAb2-AuNP-GOx). Accompanying the sandwiched immunocomplex, the labeled GOx could oxidize glucose to produce H2O2. The as-generated H2O2 could act as the coreaction reagent to trigger the chemiluminescence of the peroxyoxalate system and the PEC reaction of the BiVO4-rGO. Meanwhile, the self-illuminated light could induce photovoltaic material (BiVO4-rGO) to produce a voltage that was utilized to charge an external capacitor. With the switch closed, the capacitor could discharge through the DMM and provide an instantaneous current. Different from conventional PEC immunoassays, the as-generated photoelectron was stored in the capacitor and released instantaneously to amplify the photocurrent. Under the optimal conditions, the transient current increased with the increasing target PSA concentration in the dynamic working range from 10 pg mL(-1) to 80 ng mL(-1) with a detection limit (LOD) of 3 pg mL(-1). This work demonstrated for the first time that the peroxyoxalate CL system could be used as a suitable substitute of physical light source to apply in PEC immunoassay. In addition, this methodology afforded good reproducibility, precision, and high specificity, and the method accuracy matched well with the commercial PSA ELISA kit. Importantly, the developed split-type photoelectrochemical immunoassay could not only avoid the interfering of the biomolecules relative to the photovoltaic materials but also eliminate the need of an exciting light source and expensive instrumentation, thus representing a user-friendly and low-cost assay protocol for practical utilization in quantitative low-abundance proteins.

CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle amplification and strand hybridization.

Lysozyme with a small monomeric globular enzymatic protein is part of the innate immune system, and its deficiency can cause the increased incidence of disease. Herein, we devise a new signal-enhanced fluorescence aptasensing platform for quantitative screening of lysozyme by coupling with rolling circle amplification (RCA) and strand hybridization reaction, accompanying the assembly of CdTe/CdSe quantum dots (QDs) and hemin/G-quadruplex DNzyme. Initially, target-triggered release of the primer was carried out from DNA duplex via the reaction of the aptamer with the analyte, and the released primer could be then utilized as the template to produce numerous repeated oligonucleotide sequences by the RCA reaction. Following that, the formed long-stranded DNA simultaneously hybridized with the CdTe/CdSe QD-labeled probe and hemin/G-quadruplex DNzyme strand in the system, thereby resulting in the quenching of QD fluorescent signal through the proximity hemin/G-quadruplex DNzyme on the basis of transferring photoexcited conduction band electrons of quantum dots to Fe(III)/Fe(II)-protoporphyrin IX (hemin) complex. Under optimal conditions, the fluorescent signal decreased with the increasing target lysozyme within the dynamic range from 5.0 to 500nM with a detection limit (LOD) of 2.6nM at the 3sblank criterion. Intra-assay and interassay coefficients of variation (CVs) were below 8.5% and 11.5%, respectively. Finally, the system was applied to analyze spiked human serum samples, and the recoveries in all cases were 85-111.9%.

Plasmonic Enhancement Coupling with Defect-Engineered TiO2–x: A Mode for Sensitive Photoelectrochemical Biosensing

This work demonstrates that the photoelectric response of defect-engineered TiO2-x modified with Au nanoparticles can be modulated by oxygen vacancy concentration and excitation wavelength. When strongly plasmonic Au nanoparticles are anchored to defect-engineered TiO2-x by DNA hybridization, several times plasmonic enhancement of photocurrent occurs under 585 nm excitation, and it is employed as a novel signaling mode for developing an improved photoelectrochemical sensing platform. This signaling mode combined with exonuclease III-assisted target recycling amplification exhibits excellent analytical performance, which provides a novel photoelectrochemical detection protocol.

Semiautomated Support Photoelectrochemical Immunosensing Platform for Portable and High-Throughput Immunoassay Based on Au Nanocrystal Decorated Specific Crystal Facets BiVO4 Photoanode

Photoelectrochemical (PEC) measurement has been developed rapidly for bioanalysis in recent years. However, the actual application for most existed PEC bioanalytical systems is still a challenge because the perfect solutions for sensing surface design, high-throughput detection, and portability are lacked. To successfully overcome these limitations and realize accurate, continuous screening and assessing on prognostic indicator of early stage cancer on the spot, an innovative and portable semiautomated support power-free photoelectrochemical (SP-PEC) immunosensing platform consisted with a miniature semiautomatic injection system and digital multimeter (DMM) readout is designed (prostate specific antigen, PSA, was used as the proof-of-concept analyte). Decahedral BiVO4 that decorated with Au nanocrystal on {010} facets (Au-BiVO4) by photodeposition is used as the photoanode materials to produce photocurrent signal under irradiation of micro laser light (5.0 w, λ ≥ 380 nm). The monoclonal anti-PSA capture antibody (mAb1)-functionalized Fe3O4 magnetic nanobeads (mAb1-MN) and glucose oxidase (GOx)/monoclonal detection antibody (mAb2)-conjugated gold nanoparticle (GOx-AuNP-mAb2) are employed as immunosensing probe and signal probe, respectively. The H2O2 as an excellent holes scavenger that in suit generated from GOx oxidization glucose substrate significantly amplifies the photocurrent. The variation of instantaneous current value that registered as the signal of the immunoassay increases linearly with the logarithm of target PSA concentration increasing in a wide range from 10 pg mL-1 to 100 ng mL-1 with a low detection limit (LOD) of 4.0 pg mL-1. The SP-PEC immunosensing platform not only simplifies the assay process, but also improves detecting efficiency. The semiautomatic and portable SP-PEC analysis device allows analysis on spot and high-throughput continuous detection. Additional, we also gain deep insight into the relations between the specific shape as well as Au nanocrystal decoration and PEC activity and speculate the possible enhancement mechanisms of Au-BiVO4. Therefore, the present work not only develops a flexible SP-PEC biosensor platform for rapid and continuous detection, but also provides a possible route for designing high performance photoelectric materials.

Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion

A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.

In Situ Generation of Electron Donor to Assist Signal Amplification on Porphyrin-Sensitized Titanium Dioxide Nanostructures for Ultrasensitive Photoelectrochemical Immunoassay

An ultrasensitive photoelectrochemical (PEC) immunoassay protocol for quantitative detection of low-abundant proteins at a low potential was designed by utilizing porphyrin-sensitized titanium dioxide (TiO2) nanostructures. Experimental results demonstrated that the water-soluble 5,10,15,20-tetra(4-sulfophenyl)-21H,23H-porphyrin (TSPP) could be bound onto titanium dioxide via the sulfonic group. TSPP-sensitized TiO2 nanostructures exhibited better photoelectrochemical responses and stability in comparison with TiO2 nanoparticles alone under continuous illumination. Using carcinoembryonic antigen (CEA) as a model analyte, a typical PEC immunosensor by using TSPP-TiO2 as the affinity support of anti-CEA capture antibody (Ab1) to facilitate the improvement of photocurrent response was developed. Bioconjugates of secondary antibody and glucose oxidase with gold nanoparticles (Ab2/GOx-AuNPs) was introduced by an antigen-antibody immunoreaction. AuNP acted as a powerful scaffold to bind with bioactive molecules, while GOx catalyzed glucose to in situ generate hydrogen peroxide (H2O2). The generated H2O2 as a sacrificial electron donor could be oxidized by the photogenerated holes to assist the signal amplification at a low potential under light excitation, thus eliminating interference from other species coexisting in the samples. Under optimal conditions, the PEC immunosensor showed a good linear relationship ranging from 0.02 to 40 ng mL(-1) with a low detection limit of 6 pg mL(-1) CEA. The precision, reproducibility, and specificity were acceptable. In addition, the method accuracy was also evaluated for quantitatively monitoring human serum samples, giving results matching with the referenced CEA ELISA kit.

Cobalt-Porphyrin-Platinum-Functionalized Reduced Graphene Oxide Hybrid Nanostructures: A Novel Peroxidase Mimetic System For Improved Electrochemical Immunoassay

5,10,15,20-Tetraphenyl-21H,23H-porphine cobalt flat stacking on the reduced graphene oxide with platinum nanoparticles (PtNPs/CoTPP/rGO) were first synthesized and functionalized with monoclonal rabbit anti-aflatoxin B1 antibody (anti-AFB1) for highly efficient electrochemical immunoassay of aflatoxin B1 (AFB1) in this work. Transmission electron microscopy (TEM), atomic force microscope (AFM) and spectral techniques were employed to characterize the PtNPs/CoTPP/rGO hybrids. Using anti-AFB1-conjugated PtNPs/CoTPP/rGO as the signal-transduction tag, a novel non-enzymatic electrochemical immunosensing system was designed for detection of target AFB1 on the AFB1-bovine serum albumin-functionalized sensing interface. Experimental results revealed that the designed immunoassay could exhibit good electrochemical responses for target analyte and allowed the detection of AFB1 at a concentration as low as 5.0 pg mL−1 (5.0 ppt). Intra- and inter-assay coefficients of variation were below 10%. Importantly, the methodology was further validated for analyzing naturally contaminated or spiked blank peanut samples with consistent results obtained by AFB1 ELISA kit, thus providing a promising approach for quantitative monitoring of organic pollutants.

Reduced graphene oxide-functionalized FeOOH for signal-on photoelectrochemical sensing of prostate-specific antigen with bioresponsive controlled release system

A new and signal-on photoelectrochemical (PEC) sensing platform was successfully designed for the sensitive detection of prostate-specific antigen (PSA), using reduced graphene oxide- functionalized iron oxyhydroxide (FeOOH-rGO) as the photoactive material, accompanying target-responsive controlled release system to achieve the signal amplification. Introduction of rGO as electron mediator greatly facilitated the electron transfer from FeOOH to electrode under visible light, which inhibited the electron-hole recombination to enhance the photo-activity of FeOOH-rGO. Additionally, the bioresponsive release system was controlled via the reaction of target PSA with the aptamer capped glucose-loading mesoporous silica nanoparticle (MSN) to release numerous glucose molecules (as the electron donors) for the amplification of the photocurrent generated from FeOOH-rGO. Thus, more glucose molecules could be released and enhanced photocurrents could be obtained with the increasing PSA concentrations. Experimental results showed that the photocurrents of the PEC sensing platform were linearly dependent on the logarithm of PSA concentrations from 1.0pg/mL to 100ng/mL. Moreover, the PEC sensing system afforded good stability and specificity, and its accuracy matched well with the commercial PSA enzyme-linked immunosorbent assay (ELISA) kit. The excellent performance of the PEC sensing platform indicated its promising prospect as a useful tool for PSA detection in practical application.