Enzyme-based Signal Amplification Research Articles

Development of a colloidal gold immunochromatographic strip with enhanced signal for the detection of bovine parvovirus.

Bovine parvovirus (BPV) is a pathogen responsible for respiratory and digestive tract symptoms in calves and abortion and stillbirth in pregnant cows. In this study, we developed a colloidal gold immunochromatographic (GICG) strip with an enhanced signal for detecting BPV according to the double-antibody sandwich principle and an enzyme-based signal amplification system to amplify the signal. This system utilizes horseradish peroxidase reacting with a substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain insoluble blue products on the test and control lines. We optimized different reaction conditions, including the amount of monoclonal antibodies (mAbs), pH of the colloidal gold solution, coating solution, blocking solution, sample pad treatment solution, antibody concentration in the control line, and antibody concentration in the detection line. The sensitivity of the signal-enhanced GICG strip showed that the minimum amount for detecting BPV was 102 TCID50, 10 times higher than that of the traditional GICG strip. The results of the specificity test showed that the signal-enhanced GICG strip had no cross-reactivity with BRV, BVDV, or BRSV. The results of the repeatability test showed that the coefficient of variation between and within batches was less than 5%, showing good repeatability. Moreover, for validation, PCR and the signal-enhanced GICG strip were used to detect 280 clinical bovine fecal samples. The concordance rate compared with PCR was 99.29%. Hence, the developed strip exhibited high sensitivity and specificity for the detection of BPV. Therefore, this strip could be a rapid, convenient, and effective method for the diagnosis of BPV infection in the field.

All-in-one calcium nanoflowers for dual outputs biosensor: A simultaneous strategy for depression drug evaluation and non-invasive stress assessment

Although enzyme-based signal amplification has been well developed for biosensors, their application in low-abundance biomarker under complicated conditions detection remains challenge. Cortisol is a steroid hormone and a quantitative evaluation of cortisol can objectively assess stress and depression. However, various factors can induce slight cortisol changes in body fluids, and this in turn sets a strict requirement for bedside testing of cortisol for evaluation of stress. Herein, all-in-one calcium nanoflowers (CaHPO4-AM-HRP-SA NFs) integrated with horseradish peroxidase (HRP), α-amylase (α-AM), and streptavidin (SA) have been synthesized to develop a simple but powerful biosensor for cortisol detection. High specific surface area and allosteric modulator provided by the hybrid nanoflowers as inherent advantages significantly boosted the catalytical ability and stability compared with the free enzymes. CaHPO4-AM-HRP-SA NFs also endowed the sensor with two output signals of one sample, leading the as-prepared sensor to realize self-calibration detection. Aside from using a traditional microplate reader to measure the signal, it could also be read out by a handheld blood glucose meter and a mobile phone. The sensor exhibited attractive simplicity and sensitivity with a low LOD of 98.5 pg mL−1. It accomplished the sensitive evaluation of cortisol in rat serum and assessed the antidepressant effects of different medications. The non-invasive and reliable cortisol detection is also achieved in human urine and saliva samples. Overall, we have demonstrated that the sensor can be deployed as a promising platform to evaluate drug efficiency and monitor stress in a simple and non-invasive manner.

Usefulness of enzyme-free and enzyme-resistant detection of complement component 5 to evaluate acute myocardial infarction

Biomedical kits of simple design, low cost and high reliability are essential for the future development of point-of-care medicine, and reducing the complexity of a biosensing design can greatly lower its cost while improving its robustness. This work reports our effort towards this direction: the traditional antibody-based target recognition and enzyme-based signal amplification are replaced with synthetic probe and metal ion-catalyzed cross-linking reaction. Specifically, we have designed a sensing method to detect complement component 5 (C5) protein, using aptamer as a molecular probe, and calcium ion catalyzed amination of carboxyl as the methods of targeting and signal generation, respectively. This design has been successfully brought to detect the C5 in blood samples from healthy volunteers, patients with unstable angina and those with acute myocardial infarction. The signal response is higher in patients with acute myocardial infarction than the other two groups, and a correlation has been detected between the signal response and cardiac remodeling and dysfunction. These results may point to the potential of this method as a fast and low-cost kit for C5 detection useful for the myocardial infarction-related scenario.

Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes

Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)4–Stav or Stav–(HRP)4, respectively) using a baculovirus-silkworm expression system. Both (HRP)4–Stav and Stav–(HRP)4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)4–Stav and Stav–(HRP)4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)4–Stav was twofold higher than that of Stav–(HRP)4, and the sensitivity of (HRP)4-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)4–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.

Nano-in-Micro Smart Hydrogel Composite for a Rapid Sensitive Immunoassay.

Immunoassays are an important tool in various bioanalytical settings, such as clinical diagnostics, biopharmaceutical analysis, environmental monitoring, and food testing. An enzyme-linked immunosorbent assay (ELISA) is usually used to amplify immunoassay signals; however, it requires labor-intensive and time-consuming procedures, which hinders its application to rapid cytokine detection. In this study, a nano-in-micro composite system, where immunosensing polystyrene beads (≈320 nm) are incorporated within a stimuli-responsive microgel matrix (≈40 µm) via microfluidics, is investigated. The intrinsic volume phase-transition change properties of the smart microgels allows an enzyme-free enhanced immunoassay, enabling instant enhancement in signal-to-noise ratios of ≈5-fold. This nano-in-micro hydrogel composite offers a simple yet highly effective method for sensitive and multiplexed cytokine analysis without complex enzyme-based signal amplification steps, greatly benefitting advanced immune medicine.

Hollow Fe3O4 Nanoparticles Assisted Signal Amplification for High-Performance Redox Molecule Catalysis toward Sensitive Electrochemical Immunoassay

In this work, we prepared hollow Fe3O4 nanoparticles (Fe3O4HNPs) as nanoelectrocatalyst to obtain a multi-functionalized redox signal tag for high-performance thionine (Thi) catalysis toward ultrasensitive protein detection. Briefly, Au-Thi-Au with good conductivity and large surface area was prepared to enhance the immobilization amount of redox molecular (Thi). Simultaneously, to improve catalytic efficiency, Au-Thi-Au (redox molecule) was further anchored on the surface of Fe3O4HNPs (nanoelectrocatalyst). Obviously, the as-prepared Au-Thi-Au@Fe3O4HNPs as signal tag combines the merits of electrochemical activity, catalytic activity, magnetic response as well as nanocarrier, which is employed in electrochemical immunoassay. Importantly, owing to the hollow structure and high activity site of Fe3O4HNPs, the signal tag of Au-Thi-Au@Fe3O4HNPs exhibits high-performance Thi catalysis even in the absence of substrate, thus, giving a highly sensitive method for electrochemical detection of alpha fetoprotein (AFP) down to the 0.033 pg mL−1. Compared with traditional enzyme-based signal amplification, the proposed immunosensor avoids the addition of any substrate in testing solution, and shows good stability, sensitivity and accuracy.

One-pot bioinspired synthesis of all-inclusive protein-protein nanoflowers for point-of-care bioassay: detection of E. coli O157:H7 from milk.

Protein-protein conjugates play a vital role in bioassays with their inherent functions of biological recognition and signal amplification. Herein, a one-pot green method for synthesis of all-inclusive protein-protein nanoflowers has been developed. The protein-protein nanoflowers integrate both essential functions of biological recognition and signal amplification, and they were used as ideal signal labels for the sensitive point-of-care detection of Escherichia coli O157:H7. Especially noteworthy, the prepared Con A-invertase-CaHPO4 hybrid nanoflowers simultaneously loaded sufficient invertase and enhanced the activity of the immobilized invertase, which fits well with the requirements of signal labels for bioassays. Due to the conversion of sucrose to glucose by invertase, Con A-invertase-CaHPO4 hybrid nanoflowers were successfully used for the reliable point-of-care detection of food pathogens by a personal glucose meter. The presented approach successfully resolved the bottleneck in preparing protein-protein conjugate-based signal labels for bioassays using enzyme-based signal amplification strategies, which holds great promise to develop on-demand protein-protein conjugates for a variety of applications extending from biosensors and biomedicine to energy, environmental monitoring and remediation.

Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay

Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb2+ as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb2+ in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays.

A highly sensitive chemiluminescence sensor for detecting mercury (II) ions: a combination of Exonuclease III-aided signal amplification and graphene oxide-assisted background reduction

In this paper, we report a highly sensitive chemiluminescence (CL) sensor for Hg2+ ions based on thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry. We designed a thymine rich oligonucleotide as a capture probe and a signal probe that includes two functional domains: a horseradish peroxidase-mimicking DNAzyme domain for the generation of CL, and a recognition domain. Graphene oxide (GO) was introduced to adsorb the signal probe via p-p interaction, which brought the DNAzyme domain and GO into close proximity and quenches CL. In the presence of Hg2+ ions, the coordination of Hg2+ with the capture probe yielded a hairpin complex, triggers cascaded strand displacement reactions and Exonuclease III-assisted signal amplifications. As a result, accumulated amounts of DNAzyme were generated and released from GO, leading to an enhanced CL signal. This strategy combines enzyme-based signal amplification and GO as a background reducer, leads to a limit of detection (LOD) of 2 nmol/L. This simple detection system provides a label-free yet sensitive approach for detection of Hg2+ ions.

Aptamer-based electrochemical assay of 17β-estradiol using a glassy carbon electrode modified with copper sulfide nanosheets and gold nanoparticles, and applying enzyme-based signal amplification

We have developed an electrochemical method for the determination of 17β-estradiol. A glassy carbon electrode was modified with a composite made from copper sulfide nanosheets, gold nanoparticles, and glucose oxidase. The copper sulfide nanosheet was prepared by a single-step hydrothermal process, and its properties were characterized by X-ray powder diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron microscopy. Finally, an estradiol-specific aptamer was assembled on the electrode. The copper sulfide nanosheet on the electrode surface acts as a relatively good electrical conductor. Glucose oxidase acts as an indicator, and the dual modification of glucose oxidase and gold nanoparticles for signal amplification. The determination of 17β-estradiol was performed by differential pulse voltammetry of glucose oxidase because the signal measured at typically −0.43 V depends on the concentration of 17β-estradiol because addition of 17β-estradiol at electrode hinders electron transfer. A linear relationship exists between the peak current and the logarithm of concentration of 17β-estradiol in the 0.5 pM to 5 nM range, with a 60 f. detection limit (at 3σ/S). The method displays good selectivity over bisphenol A, 1-aminoanthraquinone and naphthalene even if present in 100-fold concentrations.

Long-term dry storage of an enzyme-based reagent system for ELISA in point-of-care devices.

Lateral flow devices are commonly used for many point-of-care (POC) applications in low-resource settings. However, they lack the sensitivity needed for many analytes relevant in the diagnosis of diseases. One approach to achieve higher sensitivity is signal amplification, which is commonly used in laboratory assays, but uses reagents that require refrigeration and inherently requires multiple assay steps not normally compatible with POC settings. Enzyme-based signal amplification, such as the one used in ELISA, could greatly improve the limit of detection if it were translated to a format compatible with POC requirements. A signal-amplified POC device not only requires the reagents to be stored in a stable form, but also requires automation of the multiple sequential steps of signal amplification protocols. Here, we describe a method for the long-term dry storage of ELISA reagents: horseradish peroxidase (HRP) conjugated antibody label and its colorimetric substrate diaminobenzidine (DAB). The HRP conjugate retained ∼80% enzymatic activity after dry storage at 45 °C for over 5 months. The DAB substrate was also stable at 45 °C and exhibited no detectable loss of activity over 3 months. These reagents were incorporated into a two-dimensional paper network (2DPN) device that automated the steps of ELISA for the detection of a malarial biomarker. These results demonstrate the potential of enzyme-based signal amplification for enhanced sensitivity in POC devices for low resource settings.

Porous platinum nanotubes modified with dendrimers as nanocarriers and electrocatalysts for sensitive electrochemical aptasensors based on enzymatic signal amplification

A highly sensitive electrochemical aptasensor for thrombin detection is developed and demonstrated by using porous platinum nanotubes modified with polyamidoamine dendrimers as nanocarriers and electrocatalysts. The proposed strategy affords a low detection limit of 0.03 pM based on enzyme-based signal amplification.

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF)

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

DNA Nanostructure-Decorated Surfaces for Enhanced Aptamer-Target Binding and Electrochemical Cocaine Sensors

The sensitivity of aptamer-based electrochemical sensors is often limited by restricted target accessibility and surface-induced perturbation of the aptamer structure, which arise from imperfect packing of probes on the heterogeneous and locally crowded surface. In this study, we have developed an ultrasensitive and highly selective electrochemical aptamer-based cocaine sensor (EACS), based on a DNA nanotechnology-based sensing platform. We have found that the electrode surface decorated with an aptamer probe-pendant tetrahedral DNA nanostructure greatly facilitates cocaine-induced fusion of the split anticocaine aptamer. This novel design leads to a sensitive cocaine sensor with a remarkably low detection limit of 33 nM. It is also important that the tetrahedra-decorated surface is protein-resistant, which not only suits the enzyme-based signal amplification scheme employed in this work, but ensures high selectivity of this sensor when deployed in sera or other adulterated samples.