基于功能化N掺杂C3N4构建光电免疫传感器检测癌胚抗原

分别合成掺杂不同比例N的C3N4纳米材料,利用红外光谱、荧光光谱等技术筛选出光电性能最优的N掺杂C3N4,并通过透射电镜、红外光谱等进行表征。将其作为光电转换材料,在氧化铟锡(ITO)电极表面构建高性能的免疫传感器。通过抗原-抗体的高特异性结合,实现对肿瘤标志物癌胚抗原的高灵敏检测。光电流结果表明:电流响应随癌胚抗原浓度的增加而减小,并在1.0 pg/mL^10 ng/mL的范围内呈现良好线性相关性。将此方法应用于检测血清样品中的癌胚抗原浓度,检测结果与商业化的酶联免疫分析方法结果进行对比没有显著差异。     

基于脂质体信号增强癌胚抗原电化学免疫传感器的研究

研究了在玻碳电极利用巯基乙胺固定纳米金、然后纳米金固载癌胚抗体(Ab1),采用脂质体包裹电子媒介体硫堇,脂质体周围联接标记辣根过氧化物酶(HRP)的癌胚抗体(Ab2)对其传感器进行信号放大,通过循环伏安法考察了该免疫传感器的电化学特性,在优化的实验条件下,该免疫传感器的峰电流随着检测溶液中癌胚抗原(CEA)浓度的增大而增大,并在0.05～200 ng/mL CEA范围内呈现线性关系,回归方程为:Δi=0.20+0.24ρ(ng/mL);检测限为:18pg/mL(R=0.9947)。该免疫传感器可用于临床上对CEA的检测。     

基于Cu2+和双环己酮草酰二腙的比色免疫分析用于测定癌胚抗原

基于Cu²⁺与双环己酮草酰二腙(BCO)的显色体系构建比色免疫传感器，并用于癌胚抗原(CEA)定量分析，满足临床检测肿瘤的要求。在磁珠表面通过抗原抗体之间的特异性结合，构建三明治型免疫复合物，通过CuO与抗体的结合实现信号转导。酸性条件下CuO可被分解为Cu²⁺, Cu²⁺与BCO在弱碱性条件下发生络合，溶液由无色变为蓝色，且溶液吸光度值与CEA浓度的对数呈线性正相关。最佳条件下，该免疫分析法的线性检测范围为0.1～40 ng/mL，检出限为0.02 ng/mL。     

构建免标记电流型免疫传感器用于检测血清中癌胚抗原(CEA)

构建了灵敏的铁氰化钾-壳聚糖-戊二醛信号体系,并以此为信号指示剂,建立了稳定、准确的免标记电化学免疫传感器用于血清中癌胚抗原(CEA)的检测。信号体系和Nafion分别修饰于玻碳电极表面,并固定CEA抗体,分别用原子力显微镜(AFM)和循环伏安法(CV)对电极修饰过程的形貌和电化学行为进行表征。结果表明,循环伏安的电流响应值与固定在电极表面的CEA浓度直接相关,且CEA浓度的对数值在0.005~40.0 ng/mL范围内与电流的降低值呈良好的线性关系,检出限为1.23 pg/mL。方法具有良好的特异性,能准确检测血清样本中CEA的浓度。     

基于聚吡咯修饰膨胀石墨电极的甲胎蛋白电化学免疫传感器

构建了测定人血清中甲胎蛋白(AFP)的电化学免疫传感器。此免疫传感器的制备采用恒电位法在膨胀石墨(EG)电极表面合成聚吡咯(PPy),再以戊二醛(GA)作为交联剂,固定辣根过氧化酶标记的AFP抗体(HRP-anti-AFP)。此免疫传感器在含AFP的溶液中于35℃温育50 min后,再在传感器表面修饰普鲁士蓝(PB)作为电子介体,抗原抗体免疫结合产生的免疫复合物会导致HRP对PB催化氧化的效率降低。在优化条件下,AFP的浓度在0.01～300μg/L范围内呈线性关系,检出限为6.25 pg/mL(S/N=3)。这种基于PPy修饰EG电极的免疫传感器制备简单,灵敏度高且价格低廉,有望成为一次性电化学免疫传感器。     

基于离子液体-石墨烯-二茂铁的高灵敏电化学免疫传感器检测CEA

研究了在PBS缓冲介质中,一种检测癌胚抗原的新型免标记免疫电化学传感器的制备,将石墨烯、二茂铁的高效催化及壳聚糖的优良生物相容性和成膜性、离子液体的导电性等优势充分结合构建了电化学免疫传感器。通过循环伏安法及交流阻抗对修饰的电极进行表征,在最优条件下,癌胚抗原的质量浓度在0.2～50.0 ng/mL的范围内与差分脉冲伏安法峰电流呈良好的线性关系,回归方程为Δi=0.38-1.31ρ,相关系数分别为0.9967,检测限为0.06 ng/mL,该传感器可用于人血清样品的测定。     

基于纳米金/壳聚糖/石墨烯的癌胚抗原阻抗型免疫传感器

研究了在PBS缓冲介质中,一种检测癌胚抗原的新型免标记阻抗型免疫传感器的制备及应用,基于石墨烯、纳米金在玻碳电极表面组装制备传感器,通过循环伏安法、交流阻抗法对制备的传感器进行表征。在优化的实验条件下,该免疫传感器的阻抗值随着检测溶液中癌胚抗原(CEA)浓度的增大而增大,并在0.1～85 ng/mL CEA范围内呈线性关系,回归方程为△Ret=1605.55+39.26ρ;检测限为0.04 ng/mL(R=0.9992)。该免疫传感器可用于临床上对CEA的检测。     

基于二氧化硅-硫堇纳米复合物构建高灵敏癌胚抗原电流型免疫传感器

利用电沉积纳米金(AuNPs)修饰玻碳电极(GCE)表面并通过AuNPs固定癌胚抗原(CEA)的捕获抗体(Ab1),以牛血清白蛋白(BSA)封闭非特异性吸附位点;以γ-(2,3环氧丙氧)丙基三甲氧基硅烷(GPMS)作交联剂,将单分散的SiO_2纳米粒子与电子媒介体硫堇(Thi)结合成SiO_2-Thi纳米复合物,偶联辣根过氧化物酶(HRP)标记的CEA二抗(HRP-Ab2)作为电化学免疫检测信号,构建了具有信号放大效应的电流型免疫传感器并用于CEA的高灵敏检测。在CEA存在下,进行电化学酶联夹心免疫反应。在含有H2O2的磷酸盐缓冲溶液(PBS)中,标记在SiO_2-Thi纳米复合物上的HRP能催化H_2O_2氧化电子媒介体Thi,产生增强的还原峰电流,从而提高检测CEA的峰电流响应信号,进而实现对CEA的高灵敏电化学酶联夹心免疫分析。在最优实验条件下,该免疫传感器的差分脉冲伏安(DPV)还原峰电流与CEA质量浓度的对数在0.01~20ng/mL范围内呈良好的线性关系,检出限为3pg/mL(S/N=3)。该传感器对血清样品进行加标回收实验,回收率为97.3%~105.7%,可初步用于临床对CEA的检测。     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

基于纳米铂-单壁碳纳米管-壳聚糖的癌胚抗原电化学阻抗型免疫传感研究

将单壁碳纳米管-壳聚糖的复合材料(SWCNTs-CS)滴涂在玻碳电极后,采用恒电位沉积的方法在SWCNTs-CS上生成纳米铂,形成Pt NPs-SWCNTsCS复合物后用以固定癌胚抗原抗体(anti-CEA)制成免疫传感界面,在癌胚抗原(CEA)的存在下,在电极表面形成抗原-抗体免疫复合物,以[Fe(CN)6]3-/4-作为混合探针电解质,构建一种简单灵敏无标记的阻抗型免疫分析方法。随着固定CEA量增加,传感器上传递电子的能力下降导致阻抗值增大,且CEA浓度在0.010~12 ng/m L范围时,与阻抗值呈现出良好的线性关系,线性方程为△Retohm=(831.3±17.8)cCEA(ng/m L)+(885.8±95.1)(R2=0.997),检测限为4.2 pg/m L(S/N=3)。该免疫传感器具有良好的稳定性和选择性,回收率在95.0%~108.6%,可初步用于临床对CEA的检测。     

Determination of Carcinoembryonic Antigen by Surface-Enhanced Raman Spectroscopy Using Gold Nanobowl Arrays

Surface-enhanced Raman scattering (SERS) based on the double-antibody sandwich format is reported for the determination of carcinoembryonic antigen. Ordered gold nanobowl arrays were fabricated and conjugated with anticarcinoembryonic as capturing substrates, and gold nanoshells, adsorbed with 4-mercaptobenzonic acid, were modified with anticarcinoembryonic antigen as labeling tags. After the carcinoembryonic antigen was captured on ordered gold nanobowl arrays, the labeling tags were bonded to the captured carcinoembryonic antigen. The interaction of SERS substrates (ordered gold nanobowl arrays) and SERS labels (gold nanoshells) showed high sensitivity and a low detection limit for carcinoembryonic antigen. The linear dynamic range of SERS for carcinoembryonic antigen was from 5?pg/mL to 100?ng/mL with a linear relationship between carcinoembryonic antigen concentration and SERS intensity. The detection limit was 1.73?pg/mL. SERS detection may be used for other cancer biomarkers and provides potential for the clinical diagnosis of cancer biomarkers.     

Carbon Nanomaterials‐based Electrochemical Immunoassay with β‐Galactosidase as Labels for Carcinoembryonic Antigen

In this study, a novel signal‐amplified strategy for sensitive electrochemical sandwiched immunoassay of carcinoembryonic antigen (CEA) was constructed based on aminofunctionalized graphene oxide (GO‐NH₂) supported AgNPs used as catalytic labels of secondary anti‐CEA and β‐galactosidase (β‐Gal), Meanwhile, sulfhydrylation single‐wall carbon nanotubes (SWCNTs‐SH) as substrate materials embellished gold electrode through Au‐SH and connected with gold nanoparticles to form anti‐CEA/AuNPs/SWCNTs‐SH/Au sensing platform through layer‐by‐layer. In the presence of analyte CEA, a sandwich‐type immunoassay format was employed for determination of CEA by using the labeled β‐Gal toward the reduction of p‐aminophenyl galactopyranoside (PAPG) and the redox reaction of AgNPs. Under optimal conditions, the increase in the current was proportional to the concentration of CEA from 0.1 pg/mL to 200 ng/mL. The detection limit (LOD) was 0.036 pg/mL CEA at 3σ. The electrochemical immunoassay displayed an acceptable precision, selectivity, stability. Clinical serum specimens were assayed with the method, and the results were in acceptable agreement with those obtained from the referenced electrochemiluminescent method.     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for sensitive detection of squamous cell carcinoma antigen on a portable pH meter by coupling enzyme-labeled hybridization chain reaction with two alternating hairpin DNA probes for the signal amplification.     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen (SCCA; cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction (HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle (AuNP). Then, the HCR reaction was readily executed between two glucose oxidase (GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

A Novel Electrochemical Aptasensor for Carcinoembryonic Antigen Detection Based on Target‐induced Bridge Assembly

The present study describes a novel electrochemical aptasensor for detection of carcinoembryonic antigen (CEA), a key cancer biomarker. The sensing strategy relied on the CEA‐induced bridge assembly, as a physical barrier, on the surface of gold electrode, resulting in a significant increase of the sensor sensitivity. Under optimal conditions, the aptasensing platform showed a wide linear range (3 pg/mL to 40 ng/mL) and a low detection limit (0.9 pg/mL). Some possible interfering materials were also assessed and the results indicated that the designed aptasensor had good specificity toward CEA. The quantitation of CEA in the spiked human serum samples confirmed the reliability and applicability of the electrochemical aptasensor. So, the developed sensing method has a potential application in the clinical diagnosis.     

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3σ. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

A novel immunoassay using platinum nanoparticles,silver enhancement and a flatbed scanner

This study reports a potentially rapid and convenient immunoassay using antibody-platinum nanoparticle (Ab-PtNPs) conjugates as a reporter molecule and a flatbed scanner for detecting immuno-reaction and measuring of the immuno-reaction signal. This study was based on the sandwich immunoassay (three-layer format) which contained a primary antibody, test antigens and a secondary antibody. The results showed that the silver precipitation phenomenon was catalyzed by Ab-PtNPs conjugates. The changing color of reaction could easily be observed by naked eye or scanner. The silver enhancement reaction, a signal amplification method in which silver ions are reduced to silver metal, is introduced to magnify the detection signal. The relationship between sample concentration and detection signal was discussed. And the detection limit (sandwich assay) for the sample antigen was 10^-1 ng/mL. Using a flatbed scanner, Ab-PtNPs conjugates and a silver enhancement reaction, a new immunoassay is constructed.