RESUMO
This work for the first time reports on a simple and rapid colorimetric immunoassay with rapid coordination of ascorbic acid 2-phosphate (AAP) and iron (III) for determination of carcinoembryonic antigen (CEA, used as a model) by using Fe2O3 nanoparticle based-chromogenic substrate system. The signal was produced rapidly (1 min) from the coordination of AAP and iron (III) with color development of colorless to brown. TD-DFT calculation methods were employed to simulate the UV-Vis spectra of AAP-Fe2+ and AAP-Fe3+ complexes. Moreover, Fe2O3 nanoparticle could be dissolved with the aid of acid, thereby releasing free iron (III). Herein, a sandwich-type immunoassay was established based on Fe2O3 nanoparticle as labels. As target CEA concentration increased, the number of Fe2O3 labelled-antibodies (bound specifically) increased, resulting in loading more Fe2O3 nanoparticle on platform. The absorbance increased as the number of free iron (III), derived from Fe2O3 nanoparticle, increased. So, the absorbance of reaction solution is positively correlated with antigen concentration. Under optimal conditions, the current results showed good performance for CEA detection in the range 0.02-10.0 ng/mL with a detection limit of 11 pg/mL. Moreover, the repeatability, stability, and selectivity of the colorimetric immunoassay were also acceptable.
Assuntos
Antígeno Carcinoembrionário , Nanopartículas , Antígeno Carcinoembrionário/química , Ferro , Compostos Cromogênicos , Colorimetria/métodos , Imunoensaio/métodos , Limite de DetecçãoRESUMO
A flexible photoelectrochemical (PEC) biosensor is proposed for the sensitive detection of ochratoxin A (OTA) based on glucose oxidase (GOx)-encapsulated target-responsive hydrogel, using Fenton reaction-mediated in situ formation of polyaniline (PANI) as signal amplified strategy. The target-responsive DNA hydrogels with high loading capacity can carry a large amount of GOx, which not only avoids laborious labeling process but also enhances the analytical performance. Upon introduction of target molecules, the hydrogel can be opened, and multiple GOx was released, thus producing lots of H2O2 via catalytic reduction of glucose. As a component of the Fenton reagent, H2O2 can react with the Fe2+ on the graphene oxidase-PAMAM-Fe2+ (GO-PAMAM-Fe2+) to generate Fe3+ and ·OH. This in turn can oxidize aniline and generate polyaniline (PANI), resulting in the enhancement of the photocurrent signal of GO-MoS2-CdS photoelectrode. The GO-PAMAM-Fe2+ as the neighborhood component of GO-MoS2-CdS-based photoactive material not only can increase the loading amount of Fe2+, but also can inhibit the decrease of photocurrent of GO-MoS2-CdS by direct modification of Fe2+ on the photoactive material. Moreover, the high loading capacity of DNA hydrogel can efficiently promote the performance of the PEC biosensor. The PEC biosensor exhibited satisfactory analytical performance for OTA with a linear range of 0.0001-0.1 ng/mL and a low detection limit of 0.05 pg/mL. It presents recommendable specificity, stability, and practical applications. Importantly, the PEC biosensor provides a new concept for construction of PEC biosensing platform.
Assuntos
Glucose Oxidase , Hidrogéis , Peróxido de Hidrogênio , Molibdênio , Compostos de Anilina , DNARESUMO
In this work, a split-type dual-mode (colorimetric/photothermal) immunoassay method was designed for point-of-care testing (POCT) detection of mycotoxins (aflatoxin B1, AFB1 as the model analyte) in foodstuffs based on Pt supported on nitrogen-doped carbon amorphous (Pt-CN). The as-synthesized Pt-CN exhibits excellent peroxidase-mimicking activity, which can catalyze the oxidization of 3,3',5,5'-tetramethylbenzidine (TMB) into TMBox with sensitive colorimetric readout in the presence of hydrogen peroxide (H2O2). Moreover, the TMBox also serves as a near-infrared (NIR) photothermal agent to convert the colorimetric readout into heat under the irradiation of an 808 nm laser. A competitive-type immunoreaction is carried out between AFB1 and glucose oxidase (GOx)-labeled AFB1-bovine serum albumin (AFB1-BSA-GOx) conjugates. With the formation of immune complexes, the entrained GOx catalyzes the hydrolysis of glucose to generate H2O2, which further involves the Pt-CN catalyzed production of TMBox to increase colorimetric/photothermal readouts. Depending on the degree of TMB oxidation, the dual-mode immunoassay provides a linear range of 1.0 pg/mL to 10 ng/mL, with a limit of detection (LOD) of 0.22 pg/mL for the colorimetric assay and 0.76 pg/mL for the photothermal assay. Moreover, the developed method is successfully used to detect AFB1 in peanuts with acceptable accuracy compared with commercially enzyme-linked immunosorbent assay (ELISA) kits. Significantly, the photothermal readout in this method is recorded on a mobile phone device without any expensive instruments, providing an affordable and convenient tool for food safety testing.
Assuntos
Aflatoxina B1 , Colorimetria , Aflatoxina B1/análise , Complexo Antígeno-Anticorpo , Benzidinas , Carbono , Colorimetria/métodos , Glucose , Glucose Oxidase , Peróxido de Hidrogênio , Imunoensaio/métodos , Limite de Detecção , Nitrogênio , Peroxidases , Soroalbumina Bovina , PlatinaRESUMO
Nanozymes are a series of elaborately designed nanomaterials that can mimic the catalytic sites of natural enzymes for reactions. Bypassing the tedious design and preparation of nanomaterial, in this work, we report on a novel just-in-time production system of copper hexacyanoferrate nanoparticles (CHNPs), which act as an oxidase-mimicking nanozyme. This system can rapidly produce CHNPs nanozyme on demand by simply mixing Cu(II) with potassium hexacyanoferrate(III) (K3[Fe(CN)6]). It is found that once K3[Fe(CN)6] is reduced to K4[Fe(CN)6], the formation of CHNPs is inhibited. Therefore, the just-in-time production system of CHNPs was coupled with alkaline phosphatase (ALP) to construct an enzyme-controllable just-in-time production (ECJP) system, in which ALP could inhibit the production of by catalyzing the hydrolysis of ascorbic acid 2-phosphate (AAP) to generating ascorbic acid (AA). The ECJP system is then used to probe the activity of ALP by employing 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the chromogenic substrate, and a detection limit of 0.003 U L-1 was achieved. Moreover, by adapting ALP as the enzyme label, an ECJP system-based colorimetric immunoassay protocol was established for sensitive detection of aflatoxin B1 (AFB1), and a detection limit as low as 0.73 pg mL-1 was achieved. The developed immunoassay method is successfully applied to the detection of AFB1 in peanut samples. The operation of ECJP system is quite simple and the coupling of ALP with CHNPs nanozyme can arouse dual enzyme-like cascade signal amplification. So, we believe this work can offer a new perspective for the development of nanozymes-based biodetection methods and colorimetric immunoassay strategies.
Assuntos
Colorimetria , Nanopartículas , Fosfatase Alcalina , Colorimetria/métodos , Cobre , Ferrocianetos , Imunoensaio/métodos , Limite de Detecção , OxirredutasesRESUMO
In-depth exploration of the local surface plasmon resonance and piezoelectric effects associated with metal can help develop efficient biosensors. Here, we presented for the first time the localized surface plasmon resonance (LSPR) and piezoelectric effects co-enhance the construction of an efficient intra-body phase electric field for the construction of efficient photoelectrochemical (PEC) biosensors. Briefly, the LSPR enhancement and piezoelectric enhancement effects between Ag nanoparticles and the piezoelectric material NaNbO3 were investigated in a PEC biosensor system under the excitation of portable UV light. Notably, the simplified treatment of the basic building blocks of the PEC sensor, including a handheld UV flashlight instead of a physical excitation light source and a digital multimeter instead of an electrochemical workstation. The capture and immunoincubation process of target PSA occurs on separated microtiter plates and hydrogen peroxide, generated by enzyme-linked immunization, induces the directional separation of electrons and holes in the composite heterogeneous material under the excitation of light. The coupling with a digital multimeter allows for real-time monitoring of photocurrents. Further, the effect of Ag deposition on piezoelectric perovskite NaNbO3 was obtained by density functional theory (DFT) calculations. Impressively, under optimized conditions, the system exhibits an ultra-wide linear range and ultra-low detection limits for the target PSA. The system is also comparable to commercially available ELISA kits at the 95% confidence level. This work provides a novel idea of enhanced PEC biosensor for rapid and accurate detection of cancer-related proteins.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Neoplasias , Técnicas Eletroquímicas , Ouro , Humanos , Limite de Detecção , Masculino , Testes Imediatos , Antígeno Prostático Específico/análise , PrataRESUMO
Hypochlorite ions (ClO-) are widely used in bleaching agents and disinfectants. However, high concentrations of chloride species are harmful to human health. Therefore, effective methods for the detection of ClO- ions are required. In this study, using 4-fluorophthalic acid and glycine, nitrogen-fluorine co-doped carbon nanodots (N,F-CDs) were synthesized by one-pot hydrothermal synthesis for use as a fluorescent probe for the fluorometric detection of ClO- in aqueous media, based on the inhibition of n â π* transitions. The excitation and emission peak centers of the N,F-CDs are at 387 and 545 nm, respectively. The N,F-CDs show a fast quenching response (<1 min) for ClO- and can be used in a wide pH range (pH 4-13). Under optimal conditions, the fluorescence intensity decreased with increase in the ClO- concentration from 0 to 35 µM, and a low limit of detection (9.6 nM) was achieved. This probe possesses excellent selectivity and high sensitivity and was used to analyze standardized samples of piped water, achieving a satisfactory recovery. Thus, this nitrogen-fluorine co-doped nanodot probe is promising for the detection of pollutants.
RESUMO
An enzyme-linked immunoassay is described for the fluorometric determination of aflatoxin B1 (AFB1). It is based on the use of carbon dots (CDs) synthesized by using Litchi chinensis as the carbon source via a hydrothermal method. The CDs were modified with MnO2 nanosheets upon which their blue fluorescence (with excitation/emission peaks at 340/425 nm) is quenched. In the presence of AFB1, a competitive immunoreaction takes place between (1) AFB1 conjugated to bovine serum albumin and deposited in the wells of a microplate, and (2) antibody against AFB1 and labeled with alkaline phosphatase (ALP). On subsequent addition of ascorbic acid 2-phosphate, it will be hydrolyzed by ALP to form ascorbic acid and phosphate. The ascorbic acid produced reduces MnO2 nanosheets to Mn2+ ions which then are relased from the CDs. This causes the recovery of fluorescence. Under optimum conditions, fluorescence decreases linearly with increasing AFB1 concentration in the range from 1.0 ng·kg-1 to 10 µg·kg-1, and the limit of detection is 0.69 ng·kg-1. The precision of this method (expressed as RSD) is ±10.3%. The accuracy was tested by analyzing both naturally contaminated and spiked food samples, and the results were in good agreement with those obtained by the established ELISA. Graphical abstract Enzyme hydrolysate-triggered redox reaction of carbon dot-functionalized MnO2 nanosheets was developed for the fluorescence immunoassay of aflatoxin B1 in foodstuff.
Assuntos
Aflatoxina B1/análise , Carbono/química , Fluorometria/métodos , Imunoensaio/métodos , Litchi/química , Compostos de Manganês/química , Nanoestruturas/química , Óxidos/química , Arachis/química , Modelos Moleculares , Conformação Molecular , Pontos Quânticos/químicaRESUMO
The authors describe a photoelectrochemical (PEC) immunoassay for determination of aflatoxin B1 (AFB1) in foodstuff. The competitive immunoreaction is carried out on a microplate coated with a capture antibody against AFB1 using AFB1-bovine serum albumin (BSA)-liposome-coated mesoporous silica nanoparticles (MSN) loaded with L-cysteine as a support. The photocurrent is produced by a photoactive material consisting of cerium-doped Bi2MoO6. Initially, L-cysteine acting as the electron donor is gated in the pores by interaction between mesoporous silica and liposome. Thereafter, AFB1-BSA conjugates are covalently bound to the liposomes. Upon introduction of the analyte (AFB1), the labeled AFB1-BSA complex competes with the analyte for the antibody deposited on the microplate. Accompanying with the immunocomplex, the liposomes on the MSNs are lysed upon addition of Triton X-100. This results in the opening of the pores and in a release of L-cysteine. Free cysteine then induces the electron-hole scavenger of the photoactive nanosheets to increase the photocurrent. The photocurrent (relative to background signal) increases with increasing AFB1 concentration. Under optimum conditions, the photoactive nanosheets display good photoelectrochemical responses, and allow the detection of AFB1 at a concentration as low as 0.1 pg·mL-1 within a linear response in the 0.3 pg·mL-1 to 10 ng·mL-1 concentration range. Accuracy was evaluated by analyzing naturally contaminated and spiked peanut samples by using a commercial AFB1 ELISA kit as the reference, and well-matching results were obtained. Graphical abstract Schematic presentation of a photoelectrochemical immunoassay for AFB1. It is based on the use of Ce-doped Bi2MoO6 nanosheets and of liposome-coated mesoporous silica nanoparticles loaded with L-cysteine.
Assuntos
Aflatoxina B1/análise , Técnicas Biossensoriais/métodos , Cisteína/química , Imunoensaio/métodos , Lipossomos/química , Nanopartículas/química , Dióxido de Silício/química , Animais , Arachis/química , Bovinos , Eletroquímica , Modelos Moleculares , Conformação Molecular , Processos Fotoquímicos , Porosidade , Soroalbumina Bovina/químicaRESUMO
A novel magnetic controlled photoelectrochemical (PEC) sensing system was designed for sensitive detection of prostate-specific antigen (PSA) using reduced graphene oxide-functionalized BiFeO3 (rGO-BiFeO3) as the photoactive material and target-triggered hybridization chain reaction (HCR) for signal amplification. Remarkably enhanced PEC performance could be obtained by using rGO-BiFeO3 as the photoelectrode material due to its accelerated charge transfer and improved the visible light absorption. Additionally, efficient and simple operation could be achieved by introducing magnetic controlled flow-through device. The assay mainly involved in anchor DNA-conjugated magnetic bead (MB-aDNA), PSA aptamer/trigger DNA (Apt-tDNA) and two glucose oxidase-labeled hairpins (H1-GOx and H2-GOx). Upon addition of target PSA, the analyte initially reacted with the aptamer to release the trigger DNA, which partially hybridized with the anchor DNA on the MB. Thereafter, the unpaired trigger DNA on the MB opened the hairpin DNA structures in sequence and propagated a chain reaction of hybridization events between two alternating hairpins to form a long nicked double-helix with numerous GOx enzymes on it. Subsequently, the enzymatic product (H2O2) generated and consumed the photo-excited electrons from rGO-BiFeO3 under visible light irradiation to enhance the photocurrent. Under optimal conditions, the magnetic controlled PEC sensing system exhibited good photocurrent responses toward target PSA within the linear range of 0.001 - 100ng/mL with a detection limit of 0.31pg/mL. Moreover, favorable selectivity, good stability and satisfactory accuracy were obtained. The excellent analytical performance suggested that the rGO-BiFeO3-based PEC sensing platform could be a promising tool for sensitive, efficient and low cost detection of PSA in disease diagnostics.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Bismuto/química , Grafite/química , Dispositivos Lab-On-A-Chip , Óxidos/química , Antígeno Prostático Específico/sangue , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Magnetismo/instrumentação , Oxirredução , Antígeno Prostático Específico/análiseRESUMO
A novel signal-amplified strategy based on dopamine-loaded liposome (DLL) was developed for competitive-type nonenzymatic photoelectrochemical (PEC) immunoassay of small- molecular aflatoxin B1 (AFB1) in foodstuff, using Mn2+-doped Zn3(OH)2V2O7·2H2O nanobelts. The signal was amplified by high-loaded capacity of liposome and the highly efficient dopamine molecule to enhance photocurrent of Mn2+-doped Zn3(OH)2V2O7·2H2O nanobelts. The loaded dopamine in the liposome was used as an electron donor to scavenge the hole and inhibit the charge recombination. To design such an immunoassay system, a AFB1-bovine serum albumin (AFB1-BSA) conjugate was covalently bound with the multifunctional liposome via the cross-linkage glutaraldehyde, whereas monoclonal anti-AFB1 antibody was labeled onto a magnetic bead by typical carbodiimide coupling. Upon addition of target AFB1, a competitive immunoreaction was carried out between the analyte and the AFB1-BSA-DLL for the conjugated antibody on the magnetic bead. Followed by magnetic separation, the carried DLL on the magnetic bead was lysed by using Triton X-100 to release the encapsulated dopamine. The as-produced dopamine (as an elector donor) increased the photocurrent of the Mn2+-doped Zn3(OH)2V2O7·2H2O nanobelts. The photocurrent depended on the as-released amount of the dopamine. The change in the photocurrent enhanced with the increasing AFB1 concentration. Under the optimal conditions, Mn2+-doped Zn3(OH)2V2O7·2H2O nanobelts exhibited good photoelectrochemical responses for the detection of AFB1 and allowed the detection of AFB1 at a concentration as low as 0.3 pg mL-1 within a linear range from 0.5 pg mL-1 to 10 ng mL-1. Importantly, this system provided an ideal PEC immune sensing platform based on Mn2+-doped Zn3(OH)2V2O7·2H2O nanobelts and the high-loaded liposome for the detection of small molecules.
Assuntos
Aflatoxina B1/análise , Técnicas Biossensoriais/métodos , Dopamina/química , Imunoensaio/métodos , Lipossomos/química , Nanoestruturas/química , Óxidos/química , Aflatoxina B1/química , Animais , Bovinos , Eletroquímica , Análise de Alimentos , Contaminação de Alimentos/análise , Limite de Detecção , Manganês/química , Processos Fotoquímicos , Soroalbumina Bovina/químicaRESUMO
A new and signal-on photoelectrochemical (PEC) sensing platform was successfully designed for the sensitive detection of prostate-specific antigen (PSA), using reduced graphene oxide- functionalized iron oxyhydroxide (FeOOH-rGO) as the photoactive material, accompanying target-responsive controlled release system to achieve the signal amplification. Introduction of rGO as electron mediator greatly facilitated the electron transfer from FeOOH to electrode under visible light, which inhibited the electron-hole recombination to enhance the photo-activity of FeOOH-rGO. Additionally, the bioresponsive release system was controlled via the reaction of target PSA with the aptamer capped glucose-loading mesoporous silica nanoparticle (MSN) to release numerous glucose molecules (as the electron donors) for the amplification of the photocurrent generated from FeOOH-rGO. Thus, more glucose molecules could be released and enhanced photocurrents could be obtained with the increasing PSA concentrations. Experimental results showed that the photocurrents of the PEC sensing platform were linearly dependent on the logarithm of PSA concentrations from 1.0pg/mL to 100ng/mL. Moreover, the PEC sensing system afforded good stability and specificity, and its accuracy matched well with the commercial PSA enzyme-linked immunosorbent assay (ELISA) kit. The excellent performance of the PEC sensing platform indicated its promising prospect as a useful tool for PSA detection in practical application.
Assuntos
Técnicas Biossensoriais , Nanopartículas/química , Antígeno Prostático Específico/isolamento & purificação , Neoplasias da Próstata/diagnóstico , Aptâmeros de Nucleotídeos/química , Preparações de Ação Retardada/química , Compostos Férricos/química , Glucose/química , Glucose/farmacologia , Grafite/química , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Dióxido de Silício/químicaRESUMO
Aflatoxin B1 (AFB1) monitoring has attracted extensive attention because food safety is a worldwide public health problem. Herein, we design a novel simultaneously visual and photoelectrochemical (PEC) immunosensing system for rapid sensitive detection of AFB1 in foodstuff. The immunoreaction was carried out on anti-AFB1 antibody-modified magnetic beads by using glucose oxidase (GOx)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags with a competitive-type immunoassay format, while the visual and PEC evaluation was performed via carbon quantum dots (CQDs)-functionalized MnO2 nanosheets. Accompanying the formation of immunocomplexes, the carried GOx initially oxidized the substrate (glucose) for the generation of H2O2, which reduced/etched MnO2 nanosheets into Mn2+ ions, thereby resulting in the dissociation of CQDs from the electrode. Within the applied potentials, the photocurrent of MnO2-CQDs-modified electrode decreased with the increasing H2O2 level in the detection cell. Meanwhile, a visual detection could be performed according to the change in the color of MnO2-CQDs-coated electrode. To elaborate, this system was aggregated into a high-throughput microfluidic device to construct a semiautomatic detection cell. Under optimal conditions, the photocurrent increased with the increasing target AFB1 within a dynamic working range from 0.01 to 20 ng mL-1 with a limit of detection (LOD) of 2.1 pg mL-1 (ppt). The developed immunoassay exhibited good reproducibility and acceptable accuracy. In addition, the method accuracy relative to AFB1 ELISA kit was evaluated for analyzing naturally contaminated or spiked peanut samples, giving the well-matched results between two methods. Although our strategy was focused on the detection of target AFB1, it is easily extended to screen other small molecules or mycotoxins, thereby representing a versatile immunosensing scheme.
Assuntos
Aflatoxina B1/análise , Técnicas Eletroquímicas/métodos , Glucose Oxidase/química , Nanopartículas Metálicas/química , Pontos Quânticos/química , Aflatoxina B1/química , Carbono/química , Glucose Oxidase/metabolismo , Imunoensaio , Luz , Limite de Detecção , Magnetismo , Compostos de Manganês/química , Óxidos/química , Reprodutibilidade dos Testes , Albumina Sérica/químicaRESUMO
This work designs a new photoelectrochemical (PEC) sensing system for the detection of carcinoembryonic antigens (CEAs) using bismuth ferrite (BiFeO3) nanostructures as photoactive materials, accompanied by the target-controlled release of glucose from multifunctional mesoporous silica nanoparticles (MSNs) for signal amplification. Glucose molecules were gated in the pores via the hybridization of a CEA aptamer with anchor DNA (aDNA, modified on the mesoporous silica nanoparticles). Upon the addition of the target CEA, the analyte competitively reacted with the aptamer to open the gate, thus resulting in the release of glucose molecules from the MSNs. Based on the oxidization of glucose in the presence of glucose oxidase, the as-generated enzymatic product (H2O2) served as an electron acceptor to enhance the photocurrent generated by the BiFeO3 nanostructures under visible light irradiation. In this way, an in situ amplified photocurrent could be achieved in that the low-concentration target CEA could cause the release of numerous glucose molecules. Experimental results suggested that the photocurrent obtained from the BiFeO3-based photoactive materials increased with increasing CEA concentration and showed a good linear dependence on the logarithm of the CEA level from 5.0 pg mL-1 to 50 ng mL-1 under optimal conditions. Additionally, the BiFeO3-based PEC sensing platform also showed good stability and favorable selectivity, and satisfactory accuracy for CEA detection in human serum specimens in comparison with a reference CEA ELISA kit. The good analytical performance of the BiFeO3-based PEC sensing method makes it a promising tool for the efficient, low-cost and convenient detection of CEAs in disease diagnosis.
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This work reports on a novel time-resolved fluorescent aptasensing platform for the quantitative monitoring of adenosine 5'-triphosphate (ATP) by interaction of dispersive/agglomerate gold nanoparticles (AuNPs) with terbium ion-coordinated carbon dots (Tb-CDs). To construct such a fluorescent nanoprobe, Tb-CDs with high-efficient fluorescent intensity are first synthesized by the microwave method with terbium ions (Tb(3+)). The aptasensing system consists of ATP aptamer, AuNP and Tb-CD. The dispersive/agglomerate gold nanoparticles are acquired through the reaction of the aptamer with target ATP. Upon target ATP introduction, the aptamers bind with the analytes to form new aptamer-ATP complexes and coat on the surface of AuNPs to inhibit their aggregation in the high salt solution. In this case, the fluorescent signal of Tb-CDs is quenched by the dispersive AuNPs on the basis of the fluorescence resonance energy transfer (FRET). At the absence of target analyte, gold nanoparticles tend to aggregate in the high salt state even if the aptamers are present. Thus, the added Tb-CDs maintain their intrinsic fluorescent intensity. Experimental results indicated that the aptasensing system exhibited good fluorescent responses toward ATP in the dynamic range from 40nM to 4.0µM with a detection limit of 8.5nM at 3sblank criterion. The repeatability and intermediate precision is less than 9.5% at three concentrations including 0.04, 0.4 and 2.0µM ATP. The selectivity was acceptable toward guanosine 5'-triphosphate, uridine 5'-triphosphate and cytidine 5'-triphosphate. The methodology was applied to evaluate the blank human serum spiked with target ATP, and the recoveries (at 3 concentration levels) ranged between 97.0% and 103.7%. Importantly, this detection scheme is rapid, simple, cost-effective, and does not require extensive sample preparation or separation.
Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Carbono/química , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/instrumentação , Térbio/química , Trifosfato de Adenosina/química , Desenho de Equipamento , Análise de Falha de Equipamento , Ouro/química , Íons/química , Nanopartículas Metálicas/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Herein, gold-silver bimetallic nanoclusters (Au-Ag NCs) with the high fluorescent intensity were first synthesized successfully and utilized for the fabrication of sensitive and specific sensing probes toward inorganic pyrophosphatase (PPase) activity with the help of copper ion (Cu(2+)) and inorganic pyrophosphate ion (PPi). Cu(2+) was used as the quencher of fluorescent Au-Ag NC, while PPi was employed as the hydrolytic substrate of PPase. The system consisted of PPi, Cu(2+) ion, and bovine serum albumin (BSA)-stabilized Au-Ag NC. The detection was carried out by enzyme-induced hydrolysis of PPi to liberate copper ion from the Cu(2+)-PPi complex. In the absence of target PPase, free copper ions were initially chelated with inorganic pyrophosphate ions to form the Cu(2+)-PPi complexes via the coordination chemistry, thus preserving the natural fluorescent intensity of the Au-Ag NCs. Upon addition of target PPase into the detection system, the analyte hydrolyzed PPi into phosphate ions and released Cu(2+) ion from the Cu(2+)-PPi complex. The dissociated copper ions readily quenched the fluorescent signal of Au-Ag NCs, thereby resulting in the decrease of fluorescent intensity. Under optimal conditions, the detectable fluorescent intensity of the as-prepared Au-Ag NCs was linearly dependent on the activity of PPase within a dynamic linear range of 0.1-30 mU/mL and allowed the detection at a concentration as low as 0.03 mU/mL at the 3sblank criterion. Good reproducibility (CV < 8.5% for the intra-assay and interassay), high specificity, and long-term stability (90.1% of the initial signal after a storage period of 48 days) were also received by using our system toward target PPase activity. In addition, good results with the inhibition efficiency of sodium fluoride were obtained in the inhibitor screening research of pyrophosphatase. Importantly, this system based on highly enhanced fluorescent Au-Ag NCs offer promise for simple and cost-effective screening of target PPase activity without the needs of sample separation and multiple washing steps.
Assuntos
Fluorescência , Ouro/química , Nanopartículas Metálicas/química , Pirofosfatases/análise , Prata/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Tamanho da Partícula , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Fluoreto de Sódio/farmacologia , Propriedades de SuperfícieRESUMO
Mycotoxins, highly toxic secondary metabolites produced by many invading species of filamentous fungi, contaminate different agricultural commodities under favorable temperature and humidity conditions. Herein, we successfully devised a novel signal-on photoelectrochemical immunosensing platform for the quantitative monitoring of mycotoxins (aflatoxin B1, AFB1, used as a model) in foodstuffs on the basis of silver nanolabels-assisted ion-exchange reaction with CdTe quantum dots (QDs) mediated hole-trapping. Initially, a competitive-type immunoreaction was carried out on a high-binding microplate by using silver nanoparticle (AgNP)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags. Then, the carried AgNPs with AFB1-BSA were dissolved by acid to release numerous silver ions, which could induce ion-exchange reaction with the CdTe QDs immobilized on the electrode, thus resulting in formation of surface exciton trapping. Relative to pure CdTe QDs, the formed exciton trapping decreased the photocurrent of the modified electrode. In contrast, the detectable photocurrent increased with the increase of target AFB1 in a dynamic working range from 10 pg mL(-1) to 15 ng mL(-1) at a low limit of detection (LOD) of 3.0 pg mL(-1) under optimal conditions. In addition, the as-prepared photoelectrochemical immunosensing platform also displayed high specificity, good reproducibility, and acceptable method accuracy for detecting naturally contaminated/spiked blank peanut samples with consistent results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.
Assuntos
Imunoensaio , Micotoxinas/análise , Pontos Quânticos/química , Prata/química , Aflatoxina B1/análise , Animais , Compostos de Cádmio/química , Bovinos , Técnicas Eletroquímicas , Eletrodos , Luz , Limite de Detecção , Nanopartículas Metálicas/química , Albumina Sérica/análise , Soroalbumina Bovina/química , Telúrio/químicaRESUMO
Herein a novel split-type photoelectrochemical (PEC) immunosensing platform was designed for sensitive detection of low-abundance biomarkers (prostate-specific antigen, PSA, used in this case) by coupling a peroxyoxalate chemiluminescence (PO-CL) self-illuminated system with digital multimeter (DMM) readout. The PEC detection device consisted of a capacitor/DMM-joined electronic circuit and a PO-CL-based self-illuminated cell. Initially, reduced graphene oxide-doped BiVO4 (BiVO4-rGO) photovoltaic materials with good photoelectric properties was integrated into the capacitor/DMM-joined circuit for photocurrent generation in the presence of hydrogen peroxide (H2O2, as the hole-trapping reagent). A sandwich-type immunoreaction with target PSA was carried out in capture antibody-coated microplates by using glucose oxidase/detection antibody-conjugating gold nanoparticle (pAb2-AuNP-GOx). Accompanying the sandwiched immunocomplex, the labeled GOx could oxidize glucose to produce H2O2. The as-generated H2O2 could act as the coreaction reagent to trigger the chemiluminescence of the peroxyoxalate system and the PEC reaction of the BiVO4-rGO. Meanwhile, the self-illuminated light could induce photovoltaic material (BiVO4-rGO) to produce a voltage that was utilized to charge an external capacitor. With the switch closed, the capacitor could discharge through the DMM and provide an instantaneous current. Different from conventional PEC immunoassays, the as-generated photoelectron was stored in the capacitor and released instantaneously to amplify the photocurrent. Under the optimal conditions, the transient current increased with the increasing target PSA concentration in the dynamic working range from 10 pg mL(-1) to 80 ng mL(-1) with a detection limit (LOD) of 3 pg mL(-1). This work demonstrated for the first time that the peroxyoxalate CL system could be used as a suitable substitute of physical light source to apply in PEC immunoassay. In addition, this methodology afforded good reproducibility, precision, and high specificity, and the method accuracy matched well with the commercial PSA ELISA kit. Importantly, the developed split-type photoelectrochemical immunoassay could not only avoid the interfering of the biomolecules relative to the photovoltaic materials but also eliminate the need of an exciting light source and expensive instrumentation, thus representing a user-friendly and low-cost assay protocol for practical utilization in quantitative low-abundance proteins.
Assuntos
Enzimas/metabolismo , Imunoensaio/métodos , EletroquímicaRESUMO
A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.
Assuntos
Cobre/metabolismo , Grafite/química , Técnicas Analíticas Microfluídicas/instrumentação , Nanoestruturas/química , Pirofosfatases/metabolismo , Cobre/química , Difosfatos/química , Difosfatos/metabolismo , Relação Dose-Resposta a Droga , Elétrons , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Íons/química , Íons/metabolismo , Fenômenos Magnéticos , Tamanho da Partícula , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/química , Fluoreto de Sódio/farmacologia , Relação Estrutura-Atividade , Propriedades de Superfície , Fatores de TempoRESUMO
In this work, a sensitive and specific electrochemical biosensor for lead ion (Pb(2+)) detection was developed by coupling with synergetic catalysis of porous Au-Pd bimetallic nanoparticles and hemin/G-quadruplex-based DNAzyme. In the presence of target Pb(2+), the substrate strand was cleaved and the enzyme strand was released. Subsequently, G-rich DNA-labeled Au-Pd bimetallic nanoparticle was linked with the released enzyme strand through the helper DNA. Upon addition of hemin, a large number of hemin/G-quadruplex-based DNAzyme molecules were formed on the electrode to serve as nicotinamide adenine dinucleotide hydrogen (NADH) oxidase and peroxidase mimics. DNAzyme could catalyzed the reduction of H2O2, generated from NADH in the presence of O2, to produce an electrochemical signal when using thionine as the electron mediator. Introduction of porors Au-Pd bimetallic nanoparticles could enhance the detectable signal and cause the increase in the sensitivity. Experimental results showed that the variations (∆I) in the cathodic peak currents of the biosensor were linearly dependent on target Pb(2+) concentrations from 1.0 pM to 100 nM with a detection limit (LOD) of 0.34 pM. The excellent performance of the sensing platform indicated its promising prospect as a valuable tool for simple and cost-effective Pb(2+) detection in practical application.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas/métodos , Chumbo/isolamento & purificação , Nanoestruturas/química , Catálise , DNA Catalítico/química , Quadruplex G , Ouro/química , Peróxido de Hidrogênio/química , Chumbo/química , Limite de Detecção , Paládio/químicaRESUMO
Biomolecular immobilization and construction of the sensing platform are usually crucial for the successful development of a high-efficiency detection system. Herein we report on a novel and label-free signal-amplified aptasensing for sensitive electrochemical detection of small molecules (adenosine triphosphate, ATP, used in this case) by coupling with target-induced hybridization chain reaction (HCR) and the assembly of electroactive silver nanotags. The system mainly consisted of two alternating hairpin probes, a partial-pairing trigger-aptamer duplex DNA and a capture probe immobilized on the electrode. Upon target ATP introduction, the analyte attacked the aptamer and released the trigger DNA, which was captured by capture DNA immobilized on the electrode to form a newly partial-pairing double-stranded DNA. Thereafter, the exposed domain at trigger DNA could be utilized as the initator strand to open the hairpin probes in sequence, and propagated a chain reaction of hybridization events between two alternating hairpins to form a long nicked double-helix. The electrochemical signal derived from the assembled silver nanotags on the nicked double-helix. Under optimal conditions, the electrochemical aptasensor could exhibit a high sensitivity and a low detection limit, and allowed the detection of ATP at a concentration as low as 0.03 pM. Our design showed a high selectivity for target ATP against its analogs because of the high-specificity ATP-aptamer reaction, and its applicable for monitoring ATP in the spiking serum samples. Improtantly, the distinct advantages of the developed aptasensor make it hold a great potential for the development of simple and robust sensing strategies for the detection of other small molecules by controlling the apatmer sequence.