Enzyme-catalyzed Precipitation Research Articles

Digital multimeter-based portable photoelectrochemical immunoassay with enzyme-catalyzed precipitation for screening carbohydrate antigen 125.

The degree of the carbohydrate antigen 125 (CA-125) level in serum is positively correlated with the severity of ovarian cancer. In this study, a facile photoelectrochemical (PEC) immunoassay was devised for sensitive detection of CA-125 employing enzyme-catalyzed precipitation to weaken the photocurrent of hollow porous In2O3 nanotubes incorporating CdS nanoparticles. Upon the addition of the target analyte, horseradish peroxidase (HRP) enriches as a result of the formation of the sandwich immunocomplex, which can catalyze the conversion of 4-chloro1-naphthol (4-CN) to benzo-4-chlorohexadienone (4-CD) employing H2O2 as a cofactor. The as-produced insoluble precipitate acts as an obstacle to hinder the absorption of visible light by photoactive materials, thereby resulting in a decrease in photocurrent. Moreover, the weakened signal can be easily read out by a digital multimeter (DMM), advancing the convenience of the detection system. The preliminary analysis data indicate that the PEC immunoassay shows an efficient response to CA-125 levels ranging from 0.1 to 100 U mL-1 with a limit of detection (LOD) as low as 0.046 U mL-1 (S/N = 3). Most importantly, the proposed portable method has shown satisfactory performance in terms of selectivity, reproducibility, stability, and analysis in complex biological matrices.

Photoelectrochemical immunoassay of alpha-fetoprotein based on a SnO2/In2S3 heterojunction and an enzyme-catalyzed precipitation strategy

We report the sensitive photoelectrochemical sandwich-type immunosensing of alpha-fetoprotein by using SnO2/In2S3 as a novel heterojunction material and horseradish peroxidase (HRP)–CeO2 as an efficient biolabel.

Tyramide signal amplification and enzyme biocatalytic precipitation on closed bipolar electrode: Toward highly sensitive electrochemiluminescence immunoassay

Abstract A novel closed bipolar electrode-based electrochemiluminescence (BPE-ECL) imaging platform for visual detection of cardiac troponin I (cTnI) was constructed using tyramine signal amplification (TSA) strategy and enzyme-catalyzed precipitation techniques (BCP). In this platform, tyramine-HRP repeats formation and the BCP were executed on the cathodic pole in the presence of H2O2, which the ECL from Ru(bpy)32+/TPA system was used as signal reporter on the anodic pole. In the presence of cTnI, a large number of HRP from HRP-Au NPs-Ab compounds and tyramine-HRP repeats catalyze the formation of insoluble precipitation on the cathodic pole of the BPE, resulting in the decreased ECL from Ru(bpy)32+/TPA system on the anodic pole. Thanks to the TSA and BCP on the cathodic pole of the BPE platform, the method for cTnI detection exhibits the detection limits of 5.0 × 10−13 g/mL by a photomultiplier tube and 5.0 × 10-12 g/mL by a charge-coupled device imaging. Furthermore, the content of cTnI in human serum samples was determined and the recovery was between 90.0 % and 112.0 %. This multiple signal amplification strategy provides new perspectives for the BPE-ECL imaging platform in the biochemical analysis of biomarkers with low abundance.

Triple sensitivity amplification for ultrasensitive electrochemical detection of prostate specific antigen

In general, current difference (ΔI) due to immunoreactions is significant in determining biosensor sensitivity. In this work, a new strategy of triple sensitivity amplification for ultrasensitive electrochemical detection of prostate specific antigen (PSA) was developed. Au-poly(methylene blue) (Au-PMB) was implemented as a redox species with strong current signal at −0.144V and used to fabricate the substrate of the biosensor. Conductive reduced graphene oxide-Au nanocomposites (Au-rGO) were coated on the Au-PMB modified glassy carbon electrode (GCE) to amplify current signal. After peptides (CEHSSKLQLAK-NH2) were fixed on the Au-rGO/Au-PMB/GCE, the fixed peptides reacted with glutaraldehyde to immobilize polydopamine-Au-horse radish peroxidase nanocomposites (PDA-Au-HRP). The electrochemical sensing interface for PSA was realized. Due to smaller resistance compared to antibodies, the peptides which can be cleaved specifically by PSA were employed. After the incubation of PSA, a large ΔI was obtained and behaved as the decrease of current signal. Then the remaining PDA-Au-HRP accelerated an enzyme-catalyzed precipitation reaction between 4-chloro-1-naphthol and H2O2. A further decrease in current signal (namely the increase in ΔI) resulted from the poorly conductive precipitation adhering onto the biosensor. The designed biosensor presented a wide linear range from 1.0fgmL−1 to 100ngmL−1 with an ultralow detection limit of 0.11fgmL−1.

DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases.

Signal enhancement strategy for a micro-arrayed polydiacetylene (PDA) immunosensor using enzyme-catalyzed precipitation

This paper describes a signal enhancement strategy to improve the sensitivity of an antibody-based immunosensor that uses polydiacetylene (PDA) liposomes to detect a target protein (human immunoglobulin E [hIgE]). To achieve ultrasensitive detection, multiple stimuli applied to PDA immunosensor chips offer a signal enhancement method that combines the primary immune reaction between antigen and antibody with the sandwich method of polyclonal antibody (pAb)-conjugated horseradish peroxidase (HRP). In the second step, fluorescence is enhanced by the mechanical pressure from the precipitate formed by enzyme catalysis. In order to detect hIgE, the surface of immobilized PDA liposomes was conjugated with monoclonal antibodies against hIgE, and fluorescence signals were detected after the antigen–antibody reaction. In this step, hIgE concentrations as low as 10ng/mL were detected. Fluorescence signals slightly increased when anti-hIgE pAb-HRP was used as an amplifying agent after primary immunoresponse. After secondary immunoresponse, HRP-catalyzed oxidation of 3,3′-diaminobenzidine produced an insoluble precipitate that strongly stimulated PDA liposomes by their weight and pressure, thereby dramatically increasing the fluorescence signal. Thus, PDA liposome immunosensor could detect hIgE concentrations as low as 0.01ng/mL, representing a 1000-fold increase in sensitivity over the signal generated by the primary immunoresponse. This study indicates that increasing the external mechanical force applied to PDA liposomes by enzyme-catalyzed precipitate formation enhanced the sensitivity of the PDA liposome immunosensor chip. This strategy can be applied to the detection of other biomolecules in experimental or clinical settings where ultrasensitive and highly specific biosensing is required.

Silica–enzyme–ionic liquid composites for improved enzymatic activity

Trypsin and pepsin enzyme-catalyzed precipitation of silica, synthesized by sol–gel chemistry in an ionic liquid, produces a composite material that demonstrates high enzymatic activity. This study investigates the structural properties of this silica–enzyme–ionic liquid composite material that allows for the retention of enzyme hydrolysis and condensation activity. The composite was prepared from a mixture of organo-functionalized triethoxysilane and tetraethoxysilane in an ionic liquid via enzyme-catalyzed direct hydrolysis and polycondensation reactions. The composite samples have been fully characterized with SEM, TEM, TG–DTA, IR, and N2 isotherms, and incorporation of the composite's organic function has been demonstrated by 29Si and 13C nuclear magnetic resonance. The presented systematic approach provides valuable information on the influence of the enzyme and ionic liquid on the properties of the silica gel and nature of the silica network as well as on the extent of enzyme and ionic liquid encapsulation. SEM and TEM reveal that the silica–enzyme–ionic liquid composites prepared from trypsin are composed of an aggregate of closely packed spherical structures ∼20 nm in diameter; however, the composites from pepsin consist of larger particles of ∼1 μm having a smooth surface. In addition, after encapsulation within the silica–ionic liquid composite, enzymes showed extremely high hydrolysis and condensation activities using trimethylethoxysilane as an enzyme substrate, and these activities were better than those of corresponding free enzyme solutions. The simple route employed can yield materials with high enzymatic activities, and this may offer very promising application prospects.

Enhanced Biomolecular Detection Based on Localized Surface Plasmon Resonance (LSPR) Using Enzyme-Precipitation Reaction

An enzyme-catalyzed precipitation reaction was employed as a means to increase the change in the LSPR signal after intermolecular bindings between antigens and antibodies occurred on gold nanodot surfaces. The gold nanodot array with an diameter of 175 nm and a thickness of 20 nm was fabricated on a glass wafer using thermal nanoimprint lithography. The human interleukin (hIL) 5 antibody was immobilized on the gold nanodot, followed by binding of hIL 5 to the anti-hIL 5. Subsequently, a biotinylated anti-hIL 5 and a alkaline phosphatase conjugated with streptavidin were simultaneously introduced. A mixture of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and nitro blue tetrazolium (NBT) was then used for precipitation, which resulted from the biocatalytic reaction of the alkaline phosphatase on gold nanodot. The LSPR spectra were obtained after each binding process. Using this analysis, the enzyme-catalyzed precipitation reaction on gold nanodots was found to be effective in amplifying the change in the peak wavelength of LSPR after molecular bindings.

Signal Amplification by Enzymatic Reaction in an Immunosensor Based on Localized Surface Plasmon Resonance (LSPR)

An enzymatic reaction was employed as a means to enhance the sensitivity of an immunosensor based on localized surface plasmon resonance (LSPR). The reaction occurs after intermolecular binding between an antigen and an antibody on gold nano-island (NI) surfaces. For LSPR sensing, the gold NI surface was fabricated on glass substrates using vacuum evaporation and heat treatment. The interferon-γ (IFN-γ) capture antibody was immobilized on the gold NIs, followed by binding of IFN-γ to the antibody. Subsequently, a biotinylated antibody and a horseradish peroxidase (HRP) conjugated with avidin were simultaneously introduced. A solution of 4-chloro-1-naphthol (4-CN) was then used for precipitation; precipitation was the result of the enzymatic reaction catalyzed the HRP on gold NIs. The LSPR spectra were obtained after each binding process. Using this method, the enzyme-catalyzed precipitation reaction on the gold NI surface was found to effectively amplify the change in the signal of the LSPR immunosensor after intermolecular binding.

Sequence-specific analysis of oligodeoxynucleotides by precipitate-amplified surface plasmon resonance measurements

A flow injection SPR spectrometer is used to detect DNA hybridization via amplification by an enzyme-catalyzed precipitation reaction. Hybridization of an oligodeoxynucleotide (ODN) target with a surface-confined ODN probe, followed by hybridization between the overhang on the resultant duplex and a biotin-tagged ODN, yields a "sandwich" complex (a three-component double-stranded). A streptavidin-horseradish peroxidase conjugate (SA-HRP) can be attached to the sandwich complex-covered surface via the biotin/streptavidin complexation. In the presence of H2O2, the HRP catalyzes oxidation of 4-chloro-1-naphthol (CN) to form a precipitate on the sensor surface. The precipitated film dramatically changes the refractive index at the metal/dielectric interface and significantly lowers the detection level. The method is shown to be reproducible, to possess high ODN sequence specificity and a high sensitivity allowing detection of ODN target concentration as low as 10 fM.

Multichannel Surface Plasmon Resonance Imaging and Analysis of Micropatterned Self-Assembled Monolayers and Protein Affinity Interactions

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-gamma antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL.

Highly sensitive amplified electronic detection of DNA by biocatalyzed precipitation of an insoluble product onto electrodes.

The amplified detection of a target DNA, based on the alkaline phosphatase oxidative hydrolysis of the soluble 5-bromo-4-chloro-3-indoyl phosphate to the insoluble indigo product as an amplification path, is addressed by two different sensing configurations. The accumulation of the insoluble product on Au electrodes or Au/quartz crystals alters the interfacial electron-transfer resistance at the Au electrode or the mass associated with the piezoelectric crystal, thus enabling the quantitative transduction of the DNA sensing by Faradaic impedance spectroscopy or microgravimetric quartz crystal microbalance measurements, respectively. One sensing configuration involves the association of a complex consisting of the target DNA and a biotinylated oligonucleotide to the functionalized transducers. The binding of the avidin/alkaline phosphatase conjugate to the sensing interface followed by the biocatalyzed precipitation provides the amplification path for the analysis of the target DNA. This analysis scheme was used to sense the target DNA with a sensitivity limit that corresponds to 5 x 10(-14) M. The second amplified detection scheme involves the use of a nucleic-acid-functionalized alkaline phosphatase as a biocatalytic conjugate for the precipitation of the insoluble product. Following this scheme, the functionalized transducers are interacted with the analyzed sample that was pretreated with the oligonucleotide-modified alkaline phosphatase, followed by the biocatalyzed precipitation as the amplification route for the analysis of the target DNA. By the use of this configuration, a detection limit corresponding to 5 x 10(-13) M was achieved. Real clinical samples of the Tay-Sachs genetic disorder were easily analyzed by the developed detection routes.

Enzyme-catalyzed synthesis of hydrated calcium oxalate

Calcium oxalate hydrate was prepared by a homogeneous precipitation method using hydrolysis of oxamic acid catalyzed by enzyme. A conventional enzyme screening strategy was adopted to obtain proper hydrolase that hydrolyzes oxamic acid to oxalic acid. Dimethylformamide-water mixed solvent was used to suppress the spontaneous hydrolysis of oxamic acid. The resultant precipitates were characterized by electron microscopy, thermogravimetry, Fourier transform-IR spectroscopy and X-ray diffraction. The calcium oxalate obtained by the enzyme-catalyzed precipitation was trihydrate and fibrous with a length of 50-100 μm while that obtained by a conventional homogeneous precipitation was monohydrate and granular.

Thin Film Biosensor for Rapid Detection of mecA from Methicillin-resistant Staphylococcus aureus

The reported incidence of methicillin-resistant Staphylococcus aureus (MRSA) isolates in hospitals has increased from 2% in 1974 to 50% in 1997 (1). In addition, MRSA is emerging as a community-acquired pathogen. Resistance is most often mediated by the mecA gene, which encodes an altered penicillin-binding protein (PBP-2A) with low affinity for β-lactam antibiotics. Rapid identification of the mecA gene is important for implementation of appropriate antibiotic therapy. MRSA is identified by either culture or molecular methods. Routine culture methods require two sequential steps, one to isolate S. aureus and the second to determine antibiotic susceptibility (2). Recently, molecular methods, including PCR, branched DNA, and cycling probe assays, have been described that identify mecA sequences from individual S. aureus colonies (3)(4)(5)(6)(7). These methods are sensitive and specific, but they all require instrumentation to interpret the results. In this report, we describe a thin film biosensor for qualitative visual detection of mecA either directly from a single S. aureus colony or from a PCR amplification reaction. The technology allows direct visual detection of the interaction of target DNA sequences with complementary oligonucleotide probes immobilized to an optically coated silicon chip (8). The key assay steps for thin film formation are: (a) simultaneous hybridization of the target sequence to covalently attached surface capture probe and solution-phase biotinylated detector probes; (b) binding of anti-biotin antibody enzyme conjugate to the annealed detector probes; and (c) mass deposition through enzyme-catalyzed precipitation (Fig. 1A⇓ ). The thin film alters the interference pattern of light on the surface, producing a perceived color change. Composition of the optical layers is designed such that small …