Highly Sensitive Electrochemical Immunosensor Based on Mesoporous PdPt Nanoparticles Anchored on a Three-dimensional PANI@CNTs Network as Nanozyme Labels

In this study, a novel strategy to amplify electrochemical signals by mesoporous PdPt nanoparticles with core-shell structures anchored on a three-dimensional PANI@CNTs network as nanozyme labels (PdPt/PANI@CNTs) was proposed for the sensitive monitoring of α-fetoprotein (AFP, Ag). First, the mesoporous PdPt nanoparticles prepared by a facile chemical reduction method had excellent biocompatibility with biomolecules, which could capture a large amount of AFP-Ab₂ (Ab₂) and exhibit plentiful pores to entrap more thionine (Thi) into mesoporous PdPt nanoparticles with enhanced loading and abundant active sites. Furthermore, the resulting mesoporous PdPt nanoparticles were abundantly dotted on the surface of a three-dimensional PANI@CNTs network with excellent conductivity and a high specific surface area through the bonding of the amino group to form PdPt/PANI@CNTs nanozyme labels. Most importantly, the as-prepared PdPt/PANI@CNTs nanozyme labels exhibited unexpected enzyme-like activity towards the reduction of hydrogen peroxide owing to the highly indexed facets, enhancing the current response to realize signal amplification. In view of the advantages of nanozyme labels and the involvement of gold nanoparticles (AuNPs, which behave as electrode materials) for the sensitive determination of AFP, the as-developed immunosensor could obtain a dynamic working range of 0.001 ng mL^−1–100.0 ng mL⁻¹ at a detection limit of 0.33 pg mL⁻¹ via DPV (at 3σ). Furthermore, the nanozyme-based electrochemical immunosensor exhibited remarkable analytical performance, which brought about feasible ideas for disease diagnosis in the future.     

以聚硫堇为电化学探针的非标记型核酸适配体传感器

采用电聚合法制备了聚硫堇氧化还原电化学探针, 以金纳米粒子为固定核酸适配体的载体构建了非标记型核酸适配体传感器. 用电化学阻抗谱对传感器的组装过程进行了监测, 用循环伏安法和差分脉冲伏安法考察了传感器的电化学行为. 结果表明, 该传感器对凝血酶的检测在1.0 pg/mL～500 ng/mL范围内呈良好的线性关系, 相关系数为0.998, 检出限为0.38 pg/mL. 该传感器制备简单、 灵敏度高且抗干扰能力强.     

A novel aptasensor based on 3D-inorganic hybrid composite as immobilized substrate for sensitive detection of platelet-derived growth factor

A novel electrochemical detection approach for platelet-derived growth factor(PDGF) via "sandwich"structure is reported in this paper. 3D-4MgCO_3 Mg(OH)_2 4H_2O-Au NPs inorganic hybrid composite was utilized as immobilized substrate for sensitive PDGF detection and Pt-Au bimetallic nanoparticles were labelled on PDGF aptamer to indirectly detect PDGF for the first time. The proposed aptasensor exhibited a high catalytic efficiency towards reduction of H_2O_2, hence the sensitive detection of PDGF was achieved.Results showed that the aptasensor exhibited excellent linear response to PDGF, in the range of 0.1 pg/m L–10 ng/m L(4 fmol/L–400 pmol/L), with detection limit of 0.03 pg/m L(1.2 fmol/L).     

A simple double-bead sandwich assay for protein detection in serum using UV-vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)⁻¹ and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.     

Portable smartphone-based microfluidic electrochemical immunosensor for ultrasensitive detection of alpha-fetoprotein in human serum

Rapid and accurate tracing of biomarkers is essential for early detecting and diagnosing of cancer. Therefore, a valid and convenient strategy needs to be developed for efficient monitoring of cancer biomarkers. Herein, we constructed a portable microfluidic electrochemical immunosensor based on three-dimensional reduced graphene oxide (3D rGO) doped with gold nanoparticles (Au NPs) for ultrasensitive determination of alpha-fetoprotein (AFP). The designed microfluidic chip, with the advantages of small injection volume, detachable structure and high integration, was fabricated by 3D printing, which only needed 9 μL of reagent to realize the high sensitivity detection. In addition, the 3D Au NPs-rGO composites with high specific surface area and electrons transfer capacity can effectively increase electroactive sites and enhance electrochemical signals. Benefiting from these features, the 3D Au NPs-rGO microfluidic electrochemical immunochip showed a wide detection range between 0.1 pg/mL–200 ng/mL and a best detection limit of 0.045 pg/mL with the high sensitivity of 175.008 μA (ng/mL)⁻¹ cm⁻². Meanwhile, the proposed immunosensor exhibited reliable AFP detection in human serum samples, which demonstrated that this portable smartphone-based microfluidic electrochemical immunosensor hold great promises in clinical detection and huge potential in personalized healthcare.     

A combination of dispersive liquid–liquid microextraction and surface plasmon resonance sensing of gold nanoparticles for the determination of ziram pesticide

We have developed a reliable, fast, and highly sensitive analytical method utilizing dispersive liquid–liquid microextraction and gold nanoparticles probes for ziram (zinc bis(dimethyldithiocarbamate)) determination. The method is based on the in situ formation of gold nanoparticles in carbon tetrachloride as an organic phase. It was found that the trace levels of ziram influenced the formation of gold nanoparticles, leading to absorbance change of a sedimented phase. The results of the colorimetric ziram determination were in the concentration range of 0.12–2.52 ng/mL with a limit of detection of 0.06 ng/mL. The formation of the stable and dispersed gold nanoparticles in the organic phase provides a good precision for dispersive liquid–liquid microextraction method, resulting in the relative standard deviation of 3.8 and 1.2% for 0.56 and 1.58 ng/mL of ziram, respectively. This method has been successfully used for the ziram determination in samples of well and river water, soil, potato, carrot, wheat, and paddy soil.     

聚多巴胺仿生纳米微球用于构建电化学免疫传感器

利用多巴胺仿生聚合方法制备了具有良好生物相容性的聚多巴胺纳米微球,并在其表面原位合成银纳米颗粒.复合物微球具有良好的催化还原H2O2的性能以及良好的结合生物分子的能力.将制备的复合物微球作为标记物,将氨基化石墨烯作为基底材料,构建了检测人免疫球蛋白(Ig G)的夹心型电化学免疫传感器.运用循环伏安法和计时电流法对构建的电化学免疫传感器进行了性能分析,并对实验条件进行了考察优化.在最佳的实验条件下,免疫传感器的线性范围是0.1 pg/m L~15 ng/m L,检出限为0.025 pg/m L.     

Electrochemical Detection of β-Lactoglobulin Allergen Using Titanium Dioxide/Carbon Nanochips/Gold Nanocomposite-based Biosensor

A label-free electrochemical immunosensor was developed for the ultra-sensitive detection of β-lactoglobulin (β-LG). The novel nanocomplex of carbon nanochips, colloidal gold nanoparticles and titanium dioxide nanoparticles TiO₂/CNC/AuC were constructed on conducting polymer, chitosan, and were characterised using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). This nanocomplex interface was studied using cyclic voltammetry (CV) and showed great improvement at the gold electrode surface with enhanced electrochemical performance, sensitivity and selectivity for β-lactoglobulin. Under optimal parameters, the square wave voltammetry (SWV) response curve was determined from 0.01 pg/mL to 500 pg/mL using [Fe(CN)₆]^3−/4−] redox probe. The calibration plot illustrates a linear relationship between log β-LG concentration and SWV current, with the limit of detection determined to be 0.01 pg/mL. This immunosensor displayed high sensitivity, selectivity, reproducibility and stability, and can be utilised for the detection of β-LG in real food samples.     

磁珠负载的适配体修饰纳米金探针用于血清中细菌内毒素检测

生物传感检测血清中的细菌内毒素具有重要的应用价值,但其开发过程受到血清所含物质复杂性的限制。本文开发了一种基于磁珠-纳米金-适配体(MB-AuNPs-APT)的生物探针比色传感器。通过生物探针捕获内毒素来影响其过氧化物酶活性,进而影响H_2O_2催化3,3′,5,5′-四甲基联苯胺(TMB)产生氧化TMB的量,最终达到比色分析内毒素的目的。分析了该方法对血清中内毒素检测的可行性,结果表明,基于MB-AuNPs-APT生物探针比色传感法的检测限为0.402ng/mL,线性范围为0.1～100 ng/mL。在50%血清样品中此生物传感器检测内毒素的回收率为99.59%～112.00%,展示了该生物探针在血清中检测内毒素的可靠性。     

A competitive microcystin-LR immunosensor based on Au NPs@metal-organic framework (MIL-101)

A porous metal organic frameworks (MOFs) material (MIL-101) based on trivalent chromium skeleton were synthesized by hydrothermal synthesis method, and loaded with Au nanoparticles (Au NPs) to prepare Au NPs@MIL-101 composite materials which were used as a marker to label anti microcystin-LR (Anti-MC-LR). The composite materials have strong catalytic properties to the oxidation of ascorbic acid. Anti-MC-LR was immobilized on glassy carbon electrode surface using electrodeposition graphene oxide (GO) as a fixed matrix to construct a competitive microcystin-LR immunosensor.     

pH Readout enhanced ELISA for point-of-care testing of cardiac troponin I

In this study, we described a pH ELISA using synthetic melanin nanoparticles (SMNPs) for the co-immobilization of glucose oxidase and second antibody as signal labels, portable pH meter as signal readout device for detecting biomarker of myocardial injury. This assay is easy-to-use, portable, sensitive and able to realize point-of-care testing (POCT), which was demonstrated the signifi cant promising in the early diagnosis and screening of acute myocardial infarction.     

A Novel Magnetism‐assisted Electrochemical Immunosensor with Sub‐Picomolar Sensitivity

A novel electrochemical immunosensor based on a magnetic glassy carbon electrode (MGCE) was developed for the quantitative determination of human immunoglobulin G (IgG). The immunosensing interface was fabricated by initially depositing silver nanoparticles on the MGCE surface and then immobilizing anti‐human IgG antibodies via the magnetic force between MGCE and Fe₃O₄ nanoparticles. The antibodies were covalently bonded to the amine‐functionalized Fe₃O₄ nanoparticles. Under optimal conditions, the magnetism‐assisted immunosensor exhibited a wide linear range from 0.1 pg/mL to 1.0 µg/mL with the detection limit of 0.05 pg/mL. Furthermore, the immunosensor displayed the advantages of good reproducibility and satisfactory stability.     

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

An amplified colorimetric method has been developed for the detection of protein and cancer cells based on the assembly of nucleic acids and proteins for the first time. In this process, the assembly of nucleic acids was triggered by a biotinylated DNA strand after a sandwich immunoreaction. The biotinylated DNA strand and sandwich immunocomplex were connected by streptavidin. Then, the assembly of biotinylated bovine serum albumin (Biotin-BSA) and streptavidin-horseradish peroxidase (SA-HRP) occurred at a node of the assembled products of nucleic acids through the biotin-streptavidin reaction. Under the catalysis of horseradish peroxidase, 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized by H₂O₂ and the oxidized product was analyzed by its UV–vis absorbance signal and sensitive colorimetric detection. This colorimetric sensor could not only achieve the quantitative determination of protein by UV–vis absorbance but could also be applied for semiquantitative determination by digital visualization. Using alpha-fetoprotein (AFP) as the model target, this proposed colorimetric method showed a wide linear range from 5 pg/mL to 1 ng/mL with a detection limit of 1.95 pg/mL by the instrument, and even 5 pg/mL target protein could be distinguished simply by the naked eye. This approach was then expanded to detect cancer cells based on the recognition of folic acid receptors that were over-expressed on the cancer cells by folic acid-tethered DNA. More importantly, this strategy can be further used as a universal colorimetric method for the determination of viruses or other proteins by changing the corresponding antibodies.     

Resonance Light Scattering Method for Microdetermination of Proteins Based on Aggregation of Au Nanoparticles

By using a resonance light scattering (RLS) technique, a highly sensitive method for protein determination based on the aggregation of Au nanoparticles on protein template is described. For the Au nanoparticles of 15 nm, the detection limit of bovine serum albumin was 5.0 ng/mL and the linear range was 10–300 ng/mL. The experimental results indicated that various metal ions do not interfere with this assay. The proposed RLS assay exhibited lower variation in response signals for the same weight of different proteins and showed satisfactory results when it was used for determination of proteins in human serum.     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

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.     

基于AuNPs/PDDA-GO纳米复合物的电化学免疫传感器的构建及对SirT1蛋白的检测

基于AuNPs/PDDA-GO纳米复合物制备了一种新型电化学免疫传感器, 并将其用于SirT1的检测. 首先, 在电极表面修饰复合材料AuNPs/PDDA-GO, 然后将目标蛋白SirT1固定到修饰了AuNPs/PDDA-GO的电极表面, 再通过特异性免疫反应结合一抗(Ab₁)和辣根过氧化酶标记的二抗分子(HRP-Ab₂), 最后用示差脉冲伏安法检测电流信号, 实现了对SirT1蛋白水平的测定. 在优化的实验条件下, SirT1蛋白的浓度在0.1~100 ng/mL范围内与响应电流呈良好线性关系, 检出限为0.029 ng/mL.     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

Gold nanoparticles/carbon nanotubes/ionic liquid microsized paste electrode for the determination of cortisol and androsterone hormones

A novel nanocomposite electrode material constituted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and n-octylpyridinium hexafluorophosphate (OPPF₆) ionic liquid was prepared and checked for the development of electrochemical (bio)sensing devices. AuNPs/MWCNTs/OPPF₆ paste electrodes with micrometer dimensions (500 μm, i.d.) were constructed and applied to the determination of cortisol and androsterone hormones. Regarding cortisol determination, the microsized paste electrode was used to detect 1-naphtol generated upon addition of 1-naphthyl phosphate as enzyme substrate in the competitive immunoassay between alkaline phosphatase-labelled cortisol and cortisol. Squarewave voltammetry allowed determining the hormone within the 0.1- to 10-ng/mL linear range (r?=?0.990) with a detection limit of 15 pg/mL and a EC₅₀ value of 0.46?±?0.06 ng/mL cortisol. The method was applied to the determination of cortisol in urine and serum samples containing a certified cortisol content. Moreover, a microsized enzyme biosensor prepared by bulk modification of the AuNPs/MWCNTs/OPPF₆ electrode with the enzyme 3α-hydroxysteroid dehydrogenase was used for the determination of androsterone through the amperometric detection of reduced nicotinamide adenine dinucleotide. A calibration plot with a linear range between 0.1 and 120 μg/mL (r?=?0.993) and a limit of detection of 89 ng/mL were obtained. The biosensor was applied to the analysis of human serum spiked with androsterone at the 250 ng/mL concentration level.     

A novel electrochemical immunosensor using β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase as labels for ultrasensitive detection of alpha-fetoprotein

In this work, a novel sandwich-type electrochemical immunosensor based on host-guest interaction was fabricated for the detection of alpha-fetoprotein (AFP). Due to the large specific surface area of multiwalled carbon nanotubes and the unique supramolecular recognition ability of β-cyclodextrins, ferrocenecarboxylic acid (Fc) was incorporated into this sensor platform by host-guest interaction to generate an electrochemical signal. And β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase (GOD-CD-Ag), was used as a label to improve the analytical performance of the immunosensor by the dual amplification strategy. The obtained GOD-CD-Ag conjugates could convert glucose into gluconic acid with the formation of hydrogen peroxide (H₂O₂). And then silver nanoparticles could in situ catalyze the reduction of the generated H₂O₂, dramatically improving the oxidation reaction of Fc. The developed immunosensor shows a wide linear calibration range from 0.001 to 5.0 ng/mL with a low detection limit (0.2 pg/mL) for the detection of AFP. The method, with ideal reproducibility and selectivity, has a wide application prospect in clinical research.