Yali Yuan

Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene sheets

In this work, a sandwich-type electrochemical aptasensor for simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin is fabricated. Reduced graphene oxide sheets (rGS) are used as matrices to immobilize the redox probes, which are subsequently coated with platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGS nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bienzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGS nanocomposites as the tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized single-walled carbon nanotubes (AuNPs@SWCNTs) as the biosensor platform enhance the surface area to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results show that the multi-labeled PtNPs-redox probes-rGS nanocomposites display satisfying electrochemical redox activity and highly electrocatalytic activity of PtNPs and bienzyme, which exhibit high sensitivity for detection of proteins. The linear range of PDGF is 0.01-35 nM with a detection limit of 8 pM, while the linear ranges from 0.02 to 45 nM and a detection limit of 11 pM for thrombin are obtained.

Electrochemiluminescence of peroxydisulfate enhanced by l-cysteine film for sensitive immunoassay

A novel label free electrochemiluminescence (ECL) immunosensor based on the ECL of peroxydisulfate solution for detection of α-1-fetoprotein (AFP) has been developed. For this proposed immunosensor, L-cysteine was firstly electrodeposited on the gold electrode surface, which promoted the electron transfer and largely enhanced the ECL of peroxydisulfate solution. Subsequently, gold nanoparticles (nano-Au) were assembled onto the L-cysteine film modified electrode to improve the absorption capacity of antibody and further amplify the ECL signal. Then, antibody was immobilized onto the electrode through nano-Au. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was firstly obtained by applying the ECL of peroxydisulfate solution without conventional luminescent reagents. The AFP was determined in the range of 0.01-100 ng mL(-1), with a low detection limit of 3.3 pg mL(-1) (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application. Moreover, this work would open up a new field in the application of peroxydisulfate solution ECL for highly sensitive bioassays.

Sandwich-type electrochemiluminescence immunosensor based on Ru-silica@Au composite nanoparticles labeled anti-AFP.

A simple and sensitive sandwich-type electrochemiluminescence immunosensor for alpha-1-fetoprotein (AFP) on a gold nanoparticles (nano-Au) modified glassy carbon electrode (GCE) was developed by using Ru-silica (Ru(bpy)(3)(2+)-doped silica) doped Au (Ru-silica@Au) composite as labels. The primary antibody, anti-AFP was first immobilized on the gold nanoparticles modified electrode due to the covalent conjugation, then the antigen and the Ru-silica@Au composite nanoparticles labeled secondary antibody was conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The surfaces of Ru-silica nanoparticles were modified via the assemble of Au nanoparticles. The prepared Ru-silica@Au composite nanoparticles own the large surface area, good biocompatibility and highly effective electrochemiluminescence properties. The morphologies of the Ru-silica@Au composite nanoparticles were investigated by using transmission electronic microscope (TEM). The Ru-silica@Au composite nanoparticles labeled anti-AFP/AFP/bovine serum albumin (BSA)/anti-AFP/nano-Au modified GCE electrode was evaluated by means of cyclic voltammetry (CV) and electrogenerated chemiluminescence (ECL). The immunosensor performed high sensitivity and wide liner for detection AFP in the range of 0.05-50 ng/mL and the limit detection was 0.03 ng/mL (defined as S/N=3).

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection.

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl(4) and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol-H(2)O(2) system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL(-1) to 80 ng mL(-1) and with a detection limit of 3.3 pg mL(-1) (SN(-1)=3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.

Glucose oxidase and ferrocene labels immobilized at Au/TiO2 nanocomposites with high load amount and activity for sensitive immunoelectrochemical measurement of ProGRP biomarker

Progastrin releasing-peptide (ProGRP) is a sensitive, specific, and reliable tumor marker with small cell lung cancer (SCLC), which may indicate an early tendency of cancer metastasis, causing high mortality rate. Thus, aiming for a more convenient assay system of SCLC, a novel immunoelectrochemical measurement for sensitive detection of ProGRP was developed in this work via Au nanoparticle/graphene modified immunosensor with ferrocene and glucose oxidase-multifunctionalized Au/TiO2 nanocomposites as a trace label. At first, Au nanoparticles (nano-Au) were attached on the TiO2 nanoparticles surface by using 3-aminopropyltriethoxy silane (APTES) as linkage reagent to obtain Au/TiO2 nanocomposites (nano-Au/TiO2). Next, glucose oxidase (GOD) and ferrocene labeled secondary antibodies (Fc-Ab2) were used to bind Au/TiO2 nanocomposites with high load amount and good biological activity, and because the increased surface area and biocompatibility of nano-Au/TiO2, the electrode can provide amplified signals. On the other hand, the nano-Au functionalized graphene sheets (GS) were used for the biosensor platform for increasing the surface area as well as improving the electronic transmission rate to capture a large amount of primary antibodies (Ab1). Then in presence of glucose, amplified signals can be obtained by an electrochemical sandwich immunoassay protocol. Based on the proposed immunosensor, the current is linear with the concentration of ProGRP being within a concentration range from 10.0 to 500 pg/mL with a limit of detection down to 3.0 pg/mL (S/N=3).

Highly conducting gold nanoparticles–graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO(2) (CS-Fc+TiO(2)) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc+TiO(2) composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc+TiO(2) membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise=3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.

Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is an outstanding DNA amplification procedure, in which the reaction can accumulate 10(9) copies from less than 10 copies of input template within an hour. While the amplification reaction is extremely powerful, the quantitative detection of LAMP products is still analytically difficult. Besides, the type of targets that LAMP can detect is also less, which to some extent limited the application of LAMP. In this study, we are reporting for the first time an efficient and accurate detection system which employs the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA). Aptamers were designed as the forward outer primer to trigger the LAMP reaction, and then the LAMP amplification products were combined with a redox active molecule methylene blue (MB) and analyzed by an electrode using differential pulse voltammograms (DPV). As the reaction progresses, the MB intercalated into double-stranded regions of LAMP amplicons reduces the free MB concentration. Hence, the peak current of reaction mixture decreased with the amplification because of the slow diffusion of MB-amplified DNA complex to the electrode surface. The peak height of the current was related to the input amount of the aptamers, providing a ready means to detection the concentration of OTA. With such design, the proposed assay showed a good linear relationship within the range of 0.001-50 nM with a detection limit of 0.3 pM (defined as S/N = 3) for OTA.

Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.

Dendrimer functionalized reduced graphene oxide as nanocarrier for sensitive pseudobienzyme electrochemicalaptasensor

A novel sensitive sandwich-type pseudobienzyme aptasensor was developed by dendrimer functionalized reduced graphene oxide (PAMMA-rGO) as nanocarrier and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme. Greatly enhanced sensitivity for the target thrombin was achieved by using a dual signal amplification strategy: first, the PAMMA-rGO not only constructed an effective platform for anchoring larger amounts of electron mediator thionine (TH) and hemin/G-quadruplex bioelectrocatalytic complex with high stability and bioactivity but also accelerated the electron transfer process assisted by the conductive rGO with amplified electrochemical signal output. Second, the hemin/G-quadruplex simultaneously acting as an NADH oxidase and HRP-mimicking DNAzyme for constructing pseudobienzyme amplifying system could in situ biocatalyze formation of H₂O₂ with high local concentrations and low transfer loss that lead to obvious signal enhancements. On the basis of the dual signal amplification strategy of PAMMA-rGO and the pseudobienzyme amplifying, the developed aptasensor could respond to 0.1 pM thrombin with a linear calibration range from 0.0002 to 30.0 nM. Compared with protein enzymes assisted bienzyme aptasensor, this new aptasensor avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided ideal candidate for the development of sensitive and simple bioanalytical platform.

Graphene-promoted 3,4,9,10-perylenetetracarboxylic acid nanocomposite as redox probe in label-free electrochemical aptasensor.

Graphene/3,4,9,10-perylenetetracarboxylic acid (GPD) with three-dimensional porous structure has been successfully synthesized and served as redox probe to construct ultrasensitive electrochemical aptasensor. The GPD nanocomposite shows promoted electrochemical redox-activity of 3,4,9,10-perylenetetracarboxylic acid (PTCA) with an obvious well-defined cathodic peak from -0.7 to 0 V that never been seen from graphene or PTCA, which avoids miscellaneous redox peaks of PTCA in electrochemical characterization, offering a novel redox probe for electrochemical sensors with highly electrochemical active area and conductivity. To the best of our knowledge, this is the first study that utilizes PTCA self-derived redox-activity as redox probe in electrochemical sensors. Moreover, the interesting GPD possesses the advantages of membrane-forming property, providing a direct immobilization of redox probes on electrode surface. This simple process not only diminishes the conventional fussy immobilization of redox probes on the electrode surface, but also reduces the participation of the membrane materials that acted as a barrier of the electron propagation in redox probe immobilization. With thrombin as a model target, the redox probe-GPD based label-free electrochemical aptasensor shows a much higher sensitivity (a detection range from 0.001 nM to 40 nM with a detection limit of 200 fM) to that of analogous aptasensors produced from other redox probes.