Jiehua Lin

Sensitive amperometric immunosensor for α-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect alpha-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml(-1) with a detection limit of 0.6 ng ml(-1). The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.

A label-free immunosensor based on modified mesoporous silica for simultaneous determination of tumor markers.

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.

Sensitive immunosensor for the label-free determination of tumor marker based on carbon nanotubes/mesoporous silica and graphene modified electrode

A novel label-free immunoassay strategy for sensitive detection of α-fetoprotein (AFP) was proposed based on controlled fabrication of single-wall carbon nanotubes (CNTs) inside the channels of mesoporous silica (MPS). The silanol groups on the internal pore walls of MPS were grafted with amino groups, while the silanol groups on the external surface were blocked by trimethylchlorosilane (TMCS). Thus, CNTs and the monoclonal antibodies of AFP (anti-AFP) could be confined inside the mesopores of MPS by the covalent linking of the carboxyl and amino groups. For the preparation of immunosensing electrode, graphene sheets (GS) and anti-AFP/CNTs/TMCS-MPS were coated on the electrode surface based on layer by layer assembly. After dipping the anti-AFP/CNTs/TMCS-MPS/GS/GCE into the sample solution, the immunoconjugates formed after the immunological reaction, which resulted in the increment of spatial blocking and impedance of the immunosensing interface. Thus, the peak current decreased with the increasing concentration of AFP. CNTs inside the mesopores could promote the electron transportation through the pore channel. Meanwhile, modified GS with distinctive conduction capacity could also improve the electrochemical response. Under the optimal experimental conditions, the label-free immunosensor could detect AFP in a linear range from 0.1 to 100 ng mL(-1) with a detection limit of 0.06 ng mL(-1) (3σ).

Immunoassay channels for α-fetoprotein based on encapsulation of biorecognition molecules into SBA-15 mesopores

A novel immunoassay channeling sensor for alpha-fetoprotein (AFP) was proposed by incorporating biorecognition molecules into the mesopores of well-ordered hexagonal SBA-15. As the biosensing element, alkaline phosphatase (ALP)-labeled antibody was embedded into the mesopores with the substrate of 1-naphthyl phosphate (1-NP). The encapsulated ALP-labeled antibody retained its bioactivities on catalytic hydrolysis reaction with 1-NP and immunological reaction with antigen. Ionic liquid-chitosan hybrid was chosen to increase film adherence and prevent the leakage of the immobilized molecules. The peak current responses decreased due to the increasing spatial blocking and impedance from the formation of nonconductive immunoconjugates on the surface of the electrode. The experimental parameters were optimized including the concentrations of the immobilized 1-NP and ALP-labeled antibody, and the amounts of SBA-15 and ionic liquid for the electrode modification. Under the optimized conditions, the constructed immunosensor could detect AFP in a linear range from 1.0 to 150 ng mL(-1) with a detection limit of 0.8 ng mL(-1). It provided an alternative strategy for the detection of antigens or other proteins by the mesoporous materials.

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity.

A label-free immunosensor by controlled fabrication of monoclonal antibodies and gold nanoparticles inside the mesopores

A label-free immunosensor for the detection of α-fetoprotein (AFP) is proposed based on controlled fabrication of monoclonal antibodies of AFP (anti-AFP) and gold nanoparticles (GNPs) inside the pores of mesoporous silica (MPS). The silanol groups on the internal pore walls were grafted by aminopropyltriethoxyl silane, whereas the silanol groups on the external surface of MPS were blocked by trimethylchlorosilane (TMCS). Thus, anti-AFP and GNPs could be confined inside the mesopores of TMCS-MPS by the covalent linking with the amino groups. The prepared anti-AFP/GNPs/TMCS-MPS particles were used to modify glassy carbon electrode (GCE) to construct a label-free immunosensor. After incubating the sample AFP with the anti-AFP/GNPs/TMCS-MPS/GCE, the immunoconjugates were formed on the surface of GCE and the spatial block increased. Thus, the peak current decreased with increasing concentrations of AFP. GNPs inside the mesopores could promote the electron transportation through the pore channel. Under the optimal experimental conditions, the fabricated immunosensor could detect AFP in a linear range from 1.0 to 90 ng ml(-1) with a detection limit of 0.2 ng ml(-1) (3σ). It provided a novel alternative method for the label-free determination of other antigens.

Antifouling aptasensor for the detection of adenosine triphosphate in biological media based on mixed self-assembled aptamer and zwitterionic peptide

Direct detection of targets in complex biological media with conventional biosensors is an enormous challenge due to the nonspecific adsorption and severe biofouling. In this work, a facile strategy for sensitive and low fouling detection of adenosine triphosphate (ATP) is developed through the construction of a mixed self-assembled biosensing interface, which was composed of zwitterionic peptide (antifouling material) and ATP aptamer (bio-recognition element). The peptide and aptamer (both containing thiol groups) were simultaneously self-assembled onto gold electrode surface electrodeposited with gold nanoparticles. The developed aptasensor possessed high selectivity and sensitivity for ATP, and it showed a wide linear response range towards ATP from 0.1pM to 5nM. Owing to the presence of peptide with excellent antifouling property in the biosensing interface, the aptasensor can detect ATP in complex biological media with remarkably reduced biofouling or nonspecific adsorption effect. Moreover, it can directly detect ATP in 1% human whole blood without suffering from any significant interference, indicating its great potential for practical assaying of ATP in biological samples.

Mixed Self-assembly of Polyethylene Glycol and Aptamer on Polydopamine Surface for Highly Sensitive and Low Fouling Detection of Adenosine Triphosphate in Complex Media

Detection of disease biomarkers within complex biological media is a substantial outstanding challenge because of severe biofouling and nonspecific adsorptions. Herein, a reliable strategy for sensitive and low-fouling detection of a biomarker, adenosine triphosphate (ATP) in biological samples was developed through the formation of a mixed self-assembled sensing interface, which was constructed by simultaneously self-assembling polyethylene glycol (PEG) and ATP aptamer onto the self-polymerized polydopamine-modified electrode surface. The developed aptasensor exhibited high selectivity and sensitivity toward the detection of ATP, and the linear range was 0.1-1000 pM, with a detection limit down to 0.1 pM. Moreover, owing to the presence of PEG within the sensing interface, the aptasensor was capable of sensing ATP in complex biological media such as human plasma with significantly reduced nonspecific adsorption effect. Assaying ATP in real biological samples including breast cancer cell lysates further proved the feasibility of this biosensor for practical application.

A Label-free Immunosensor based on Ionic Liquids Modified Mesoporous Silica for Simultaneous Determination of Two Tumor Markers

A label-free electrochemical immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). The functional mesoporous silica was synthesized for the construction of the label-free immunosensor. The Si-OH groups on the external surface of the mesoporous silica were termi- nated by trimethylchlorosilane (TMCS), while the Si-OH groups on the internal pore walls were modified with amino groups. The electrochemical substrates of ferrocenecarboxylic acid (FCA) and methylene blue (MB) were co-immobilized inside the channels of the ionic liquids (ILs) modified mesoporous silica (MPS), respectively. The monoclonal antibody of CEA (anti-CEA) and the monoclonal antibody of AFP (anti-AFP) were respectively co-immobilized inside the materials of FCA-IL-MPS and MB-IL-MPS. The ITO slide was separated lengthways into two uniform parts by insulation glue so as to avoid the cross-talk of the two portions. Finally, the suspension solutions were coated respectively onto the different areas of indium-tin oxide (ITO) electrode. The double-analyte immunosensor was constructed by the probes of CEA and AFP onto the different areas of ITO. When the double-analyte immunosensor was dipped into the sample solution, the antigens of CEA and AFP reacted with their corresponding monoclonal antibodies on the different area of the modified ITO electrode. After the immunological reaction, the nonconductive immunoconjugates formed inside the MPS channels. With the increasing concentrations of CEA and AFP antigens, the spatial blocking and impedance on the sensor surface increased, thus the elec- tric response transfer from the solution to the electrode surface was blocked and the DPV currents decreased. The electro- chemical signals for CEA were detected by using FCA as the electron mediator. The electrochemical signals for AFP were detected by using MB as the electrochemical substrates. Then, the simultaneous detection of CEA and AFP could be achieved. The electrode modification process was further characterized by cyclic voltammetric measurements and electro- chemical impedance spectroscopy. IL units inside the mesopores could promote the electron transportation through the pore channel. To clarify the adsorption of FCA and MB into the mesopores of ILs-modified MPS, the IR spectra were recorded. The linear ranges of CEA and AFP were 0.5~80 ngmL -1 and 0.5~100 ngmL -1 with the detection limits of 0.1 ngmL -1