Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers

We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab(2)) linked to carbon nanotubes (CNT) at high HRP/Ab(2) ratio. This approach provided a detection limit of 4 pg mL(-)(1) (100 amol mL(-)(1)), for prostate specific antigen (PSA) in 10 microL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.     

Two‐step signal amplification for high‐sensitivity detection of biomarkers using gold nanoparticle–based conjugates

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)‐based, two‐step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96‐well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)‐conjugated detection antibodies were labeled on large‐size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high‐sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate‐specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.     

Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase

In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples Os^IIIHRP/Os^IVHRP, Os^IVHRP/Os^VHRP and Os^VHRP/Os^VIHRP. The midpoint potentials differ dependent on the electrode material used with E_1/2 (Os^III/IV) of − 0.4 V (ITO) and − 0.25 V (GC), E_1/2 (Os^IV/^V) of − 0.16 V (ITO) and + 0.10 V (GC), and E_1/2 (Os^V/VI)of + 0.18 V (ITO), respectively. Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.     

Efficient electron transport across nickel powder modified basal plane pyrolytic graphite electrode: Sensitive detection of sulfhydryl degradation products of the V-type nerve agents

This communication describes the electron transport behaviour of basal plane pyrolytic graphite electrodes (BPPGEs) modified with nickel powder (BPPGE-Ni), single-walled carbon nanotube (BPPGE-SWCNT) and BPPGE-Ni decorated with SWCNT via drop-dry method (BPPGE-Ni-SWCNT). The BPPGE-Ni gave enhanced Faradaic response for the redox probe (Ferricyanide/Ferrocyanide species) and also displayed enhanced electrocatalytic behaviour towards the detection of degradation products of V-type nerve agents, dimethylaminoethanethiol (DMAET) and diethylaminoethanethiol (DEAET) with high sensitivity (∼23 × 10⁻³ AM⁻¹) and low detection limits (4.0–9.0 μM range). When compared to the notable electrodes and detection protocols reported in the literature, BPPGE-Ni exhibits more promising features required for a simple, highly sensitive, fast and less expensive electrode for the detection of these V-type nerve agents in aqueous solution. The efficient response of the BPPGE-Ni is attributed to the high microscopic surface area of the nickel powder. The poor response of the BPPGE-Ni-SWCNT suggests that nickel impurity in the SWCNT did not show any detectable impact on its electron transfer kinetics.     

Flower-like self-assembly of gold nanoparticles for highly sensitive electrochemical detection of chromium(VI)

We report here the fabrication of a flower-like self-assembly of gold nanoparticles (AuNPs) on a glassy carbon electrode (GCE) as a highly sensitive platform for ultratrace Cr(VI) detection. Two AuNP layers are used in the current approach, in which the first is electroplated on the GCE surface as anchors for binding to an overcoated thiol sol–gel film derived from 3-mercaptopropyltrimethoxysilane (MPTS). The second AuNP layer is then self-assembled on the surface of the sol–gel film, forming flower-like gold nanoelectrodes enlarging the electrode surface. When functionalized by a thiol pyridinium, the fabricated electrode displays a well-defined peak for selective Cr(VI) reduction with an unusually large, linear concentration range of 10–1200 ng L⁻¹ and a low detection limit of 2.9 ng L⁻¹. In comparison to previous approaches using MPTS and AuNPs on Au electrodes, the current work expands the use of AuNPs to the GCE. Subsequent functionalization of the secondary AuNPs by a thiol pyridinium and adsorption/preconcentration of Cr(VI) lead to the unusually large detection range and high sensitivity. The stepwise preparation of the electrode has been characterized by electrochemical impedance spectroscopy (EIS), scanning electronic microscopy (SEM), and IR. The newly designed electrode exhibits good stability, and has been successfully employed to measure chromium in a pre-treated blood sample. The method demonstrates acceptable fabrication reproducibility and accuracy.     

An Electrochemical Impedance Immunosensor Based on Gold Nanoparticle‐Modified Electrodes for the Detection of HbA1c in Human Blood

A label‐free immunosensor for the detection of HbA1c was developed based on gold nanoparticle (AuNP)‐aryl diazonium salt modified glassy carbon (GC) electrode where transduction is achieved using electrochemical impedance spectroscopy (EIS). GC electrodes were first modified with 4‐aminophenyl (Ph‐NH₂) layers to which AuNPs were attached. Thereafter an oligo(ethylene glycol) (OEG‐COOH) species were covalently attached to the remaining free amine groups on the Ph‐NH₂ surface. The AuNP surfaces were further modified with Ph‐NH₂ followed by attachment of a glycosylated pentapeptide (GPP), an analogon to HbA1c. Exposure of this interface to anti‐HbA1c IgG resulted in a change in charge transfer resistance (R_ct) due to the anti‐HbA1c IgG selectively complexing to the surface bound GPP. To detect the amount of HbA1c, a competitive inhibition assay was employed where the surface bound GPP and HbA1c in solution compete for the anti‐HbA1c IgG antibodies. The higher the concentration of HbA1c, the less antibody binds to the sensing interface and the lower the change of R_ct. The response of the immunosensor is linear with the HbA1c% of total haemoglobin in the range of 0%–23.3%. This competitive inhibition assay can be used for the detection of HbA1c in human blood. The performance of the immunosensor for detection of HbA1c in human blood is comparable to the clinical laboratory method.     

Long Distance Electron Transfer Across >100 nm Thick Au Nanoparticle/Polyion Films to a Surface Redox Protein

Glutathione‐decorated 5 nm gold nanoparticles (AuNPs) and oppositely charged poly(allylamine hydrochloride) (PAH) were assembled into {PAH/AuNP}_n films fabricated layer‐by‐layer (LbL) on pyrolytic graphite (PG) electrodes. These AuNP/polyion films utilized the AuNPs as electron hopping relays to achieve direct electron transfer between underlying electrodes and redox proteins on the outer film surface across unprecedented distances >100 nm for the first time. As film thickness increased, voltammetric peak currents for surface myoglobin (Mb) on these films decreased but the electron transfer rate was relatively constant, consistent with a AuNP‐mediated electron hopping mechanism.     

Electrochemical Sensing Platforms for HER2‐ECD Breast Cancer Biomarker Detection

Screening and early diagnosis are crucial to increase the success of cancer patients’ treatments and improve the survival rate. To contribute to this success, distinct electrochemical immunosensing platforms were developed for the analysis of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2‐ECD) through sandwich assays on nanomaterial‐modified screen‐printed carbon electrodes (SPCEs). The most promising platforms showed to be SPCEs modified with (i) gold nanoparticles (AuNPs) and (ii) multiwalled carbon nanotubes combined with AuNPs. The antibody‐antigen interaction was detected using a secondary antibody labelled with alkaline phosphatase and 3‐indoxyl phosphate and silver ions as the enzymatic substrate. The electrochemical signal of the enzymatically generated metallic silver was recorded by linear sweep voltammetry. Under the optimized conditions, linear calibration plots were obtained between 7.5 and 50 ng/mL and the total assay time was 2 h 20 min, achieving LODs of 0.16 ng/mL (SPCE‐MWCNT/AuNP) and 8.5 ng/mL (SPCE‐AuNP), which are well below the established cut‐off value of 15 ng/mL for this cancer biomarker.     

高灵敏甲胎蛋白夹心免疫传感器的制备

构建了新型甲胎蛋白(AFP)夹心免疫传感器.采用金纳米粒子-氧化石墨烯-普鲁士蓝纳米立方体(AuNP-GO-PBNCs)纳米复合材料标记甲胎蛋白(AFP)二抗,将制备的金-聚多巴胺-四氧化三铁(Au-PDA-Fe3O4)磁性纳米复合物固定在自制的磁性电极表面,通过吸附作用固定AFP一抗,用牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点.在37℃下与AFP抗原溶液孵育50 min,最后将电极放入AuNP-GO-PBNCs纳米复合材料标记的二抗溶液中孵育,基于此建立了采用普鲁士蓝(PB)标记的的夹心免疫传感器检测AFP的方法.在最佳实验条件下,PB催化H2O2氧化的响应电流与AFP的浓度表现出两段线性关系,线性范围分别为0.005~1.000 ng/mL和1~20 ng/mL, 检出限(LOD, S/N=3)为1.0 pg/mL.本方法具有灵敏度高、选择性好的特点.     

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 μM and from 0.4 to 2.4 μM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18 ± 0.01 μM in the case of DAO/HRP and 0.40 ± 0.04 μM (α = 0.05 and β = 0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.     

A new modified conducting carbon composite electrode as sensor for ascorbate and biosensor for glucose

A new carbon-based conducting composite has been developed as electrochemical sensor and biosensor for the amperometric detection of ascorbate and glucose. Electrocatalytic oxidation of ascorbate has been done successfully at unmodified cellulose acetate-graphite composite electrodes, the sensor being highly sensitive, selective and with a low detection limit at 0.0 V vs. SCE and was successfully applied for ascorbate determination in commercial fruit juice samples. An interference free glucose biosensor has also been developed, based on the immobilisation of glucose oxidase by cross-linking with glutaraldehyde on poly (neutral red) modified composite electrodes. The biosensor exhibits a higher sensitivity of 31.5 ± 1.7 µA cm^− 2 mM^− 1 than other carbon-composite-based glucose biosensors, a detection limit of 20.3 µM and a very short response time.     

An ultrasensitive hydrogen peroxide biosensor based on electrocatalytic synergy of graphene–gold nanocomposite, CdTe–CdS core–shell quantum dots and gold nanoparticles

We first reported an ultrasensitive hydrogen peroxide biosensor in this work. The biosensor was fabricated by coating graphene–gold nanocomposite (G–AuNP), CdTe–CdS core–shell quantum dots (CdTe–CdS), gold nanoparticles (AuNPs) and horseradish peroxidase (HRP) in sequence on the surface of gold electrode (GE). Cyclic voltammetry and differential pulse voltammetry were used to investigate electrochemical performances of the biosensor. Since promising electrocatalytic synergy of G–AuNP, CdTe–CdS and AuNPs towards hydrogen peroxide was achieved, the biosensor displayed a high sensitivity, low detection limit (S/N = 3) (3.2 × 10⁻¹¹ M), wide calibration range (from 1 × 10⁻¹⁰ M to 1.2 × 10⁻⁸ M) and good long-term stability (20 weeks). Moreover, the effects of omitting G–AuNP, CdTe–CdS and AuNP were also examined. It was found that sensitivity of the biosensor is more 11-fold better if G–AuNP, CdTe–CdS and AuNPs are used. This could be ascribed to improvement of the conductivity between graphene nanosheets in the G–AuNP due to introduction of the AuNPs, ultrafast charge transfer from CdTe–CdS to the graphene sheets and AuNP due to unique electrochemical properties of the CdTe–CdS, and good biocompatibility of the AuNPs for horseradish peroxidase. The biosensor is of best sensitivity in all hydrogen peroxide biosensors based on graphene and its composites up to now.     

Synergistic effect of mediator–carbon nanotube composites for dehydrogenases and peroxidases based biosensors

Very sensitive, low cost and reliable NADH and H₂O₂ sensors were realised and used for development of enzyme based biosensors. The active surface of the electrodes was modified with a nanocomposite obtained by modification of SWNT with a proper mediator: Meldola Blue (for NADH) and Prussian Blue (for H₂O₂). Low applied potential of − 50 mV vs. Ag/AgCl reference electrode proved the synergistic effect of nanocomposite materials towards NADH and H₂O₂ detection. Biosensors for malic acid and alkylphenols have been developed, using mediator-functionalised-SWNT-based electrodes and two different classes of enzymes: NAD⁺-dependent dehydrogenases and peroxidases. Immobilization of the enzymes was realised using a series of different procedures — adsorption, Nafion membrane, sol–gel and glutaraldehyde, in order to find the best configuration for a good operational stability. A higher sensitivity comparing with other reported biosensors of about 12.41 mA/M·cm² was obtained for l-malic acid biosensor with enzyme immobilised in Nafion membrane. Phenol, 4-t-octylphenol and 4-n-nonylphenol were used as standard compounds for HRP based biosensor. Fast biosensor response and comparable detection limit with HPLC methods were achieved.     

Peroxidase activity of enzymes bound to the ends of single-wall carbon nanotube forest electrodes

This communication reports the first example, to our knowledge, of enzymes covalently attached onto the ends of vertically oriented single-wall carbon nanotube (SWNT) forest arrays used as electrodes. Quasi-reversible Fe^III/Fe^II voltammetry was observed for the iron heme enzymes myoglobin and horseradish peroxidase coupled to carboxylated ends of the nanotube forests by amide linkages. Results suggest that the “trees” in the nanotube forest behaved electrically similar to a metal, conducting electrons from the external circuit to the redox sites of the enzymes. Electrochemically manifested peroxidase activity of myoglobin and horseradish peroxidase attached to the SWNT forests was demonstrated, with detection limits for hydrogen peroxide in buffer solutions of ∼100 nM. These prototype SWNT-forest biosensors are easy to prepare, and enzyme layers were stable for weeks.     

Improved Performance of an Amperometric Biosensor with Polydiaminonaphthalene on Electrochemically Deposited Au Nanoparticles

The performance of an enzyme sensor fabricated through covalent bond formation on the HRP‐bonded poly(1,8‐diaminonaphthalene) (polyDAN) layer with gold nanoparticles (AuNPs) was applied to catalyze the electrochemical reduction of H₂O₂. The surface characteristics of the sensor probe were studied using cyclic voltammetry, SEM, XPS, QCM, and impedance spectroscopy. The AuNP‐deposited surface resulted in higher conductivity and sensitivity for H₂O₂ detection in phosphate buffer solution. A linear calibration plot was obtained in the H₂O₂ concentration range between 10.0 μM and 25.0 mM with detection limit 5.0±1.25 μM. The lifetime of HRP/polyDAN/AuNP/GC probe was over 70 days without response loss.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Mediated amperometric immunosensing using single walled carbon nanotube forests

A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes called SWNT forests. The forests were self-assembled from oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes. The nanotube forests were characterized using atomic force microscopy and resonance Raman spectroscopy. Anti-biotin antibody strongly adsorbed to the SWNT forests. In the presence of a soluble mediator, the detection limit for horseradish peroxidase (HRP) labeled biotin was 2.5 pmol ml(-1) (2.5 nM). Unlabelled biotin was detected in a competitive approach with a detection limit of 16 nmol ml(-1) (16 microM) and a relative standard deviation of 12%. The immunosensor showed low non-specific adsorption of biotin-HRP (approx. 0.1%) when blocked with bovine serum albumin. This immunosensing approach using high surface area, patternable, conductive SWNT assemblies may eventually prove useful for nano-biosensing arrays.     

Two kinds of electrochemical immunoassays for the tumor necrosis factor α in human serum using screen-printed graphite electrodes modified with poly(anthranilic acid)

We present two kinds of electrochemical immunoassays for the tumor necrosis factor α (TNF-α) which is a protein biomarker. The antibody against TNF-α was immobilized on a graphite screen-printed electrode modified with poly-anthranilic acid (ASPE). The first is based on impedimetry (and thus label-free) and the target antigen (TNF-α) is captured by the surface of the modified electrode via an immunoreaction upon which impedance is changed. This sensing platform has a detection limit of 5.0 pg mL^?1. In the second approach, the monoclonal antibodies on the modified electrode also bind to the target antigen (TNF-α), but detection is based on a sandwich immunoreaction. This is performed by first adding secondary anti-TNF-α antibodies labeled with horseradish peroxidase, and then detecting the response of the sandwich system by adding hydrogen peroxide and acetaminophen as a probe system for HRP activity. This immunosensor also has a very low detection limit (3.2 pg mL^?1). The experimental conditions of both assays were studied and optimized via electrochemical impedance spectroscopy and differential pulse voltammetry. The method was then applied to the determination of TNF-α in serum samples where it displayed high sensitivity, selectivity and reproducibility.

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Electrochemical immunosensors for interleukin-6. Comparison of carbon nanotube forest and gold nanoparticle platforms

Electrochemical immunosensors based on single wall nanotube (SWNT) forests and 5 nm glutathione-protected gold nanoparticles (GSH-AuNP) were developed and compared for the measurement of human cancer biomarker interleukin-6 (IL-6) in serum. Detection was based on sandwich immunoassays using multiple (14–16) horseradish peroxidase labels conjugated to a secondary antibody. Performance was optimized by effective blocking of non-specific binding (NSB) of the labels using bovine serum albumin. The GSH-AuNP immunosensor gave a detection limit (DL) of 10 pg mL^?1 IL-6 (500 amol mL^?1) in 10 μL calf serum, which was 3-fold better than 30 pg mL^?1 found for the SWNT forest immunosensor for the same assay protocol. The GSH-AuNPs platform also gave a much larger linear dynamic range (20–4000 pg mL^?1) than the SWNT system (40–150 pg mL^?1), but the SWNTs had 2-fold better sensitivity in the low pg mL^?1 range.