Highly sensitive determination of doxorubicin and daunorubicin based on their effect on the resonance light scattering signals of the ethidium-DNA complex

We have developed a resonance light scattering (RLS) quenching assay for the highly sensitive determination of doxorubicin (DOX) and daunorubicin (DAU). It is based on the reduction of the intensity of the shoulder of the RLS spectra at 443?nm. The intensity of the RLS of the ethidium-DNA system decrease linearly on addition of trace quantities of DOX or DAU within the concentration range of 0.008 to 12.0???g?mL^?1 for DOX, and of 0.010 to 21.0???g?mL^?1 for DAU. The detection limits are 3.0 and 5.0?ng?mL^?1, respectively. The assay was successfully applied to the determination of DAU in synthetic and serum samples. Compared to the reported methods for anthracyclines, this assay displays higher sensitivity, lower detection limits, and a wider linear range.

Electrodeposition of CdSe quantum dots and its application to an electrochemiluminescence immunoassay for α-fetoprotein

We report on the first label-free electrochemiluminescence (ECL) immunosensor for α-fetoprotein (AFP). It is based on the use of CdSe quantum dots that were electrodeposited directly on a gold electrode from an electrolyte (containing cadmium sulfate, EDTA and selenium dioxide) by cycling the potential between 0 and -1.2?V (vs. SCE) for 60?s. The electrodeposited dots were characterized by scanning electron microscopy and energy dispersive spectroscopy. Under optimal conditions, the specific immunoreaction between AFP and anti-AFP resulted in a decrease of the ECL signal because of the steric hindrance and the transfer inhibition by peroxodisulfate. The quenching effect of the immunoreaction on the intensity of the ECL was used to establish a calibration plot which is linear in the range from 0.05 to 200?ng?mL^?1. The detection limit is 2?pg?mL^?1. The assay is highly sensitive and satisfactorily reproducible. In our opinion it opens new avenues to apply ECL in label-free biological assays.

Interaction of polymyxin B with ds-DNA, and determination of DNA or polymyxin B via resonance Rayleigh scattering and resonance non-linear scattering spectra

Assays were developed for DNA or polymyxin B (PMB) based on enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (including second order scattering and frequency doubling scattering) that result from the interaction of PMB with DNA. A minor-groove binding mechanism is suggested from the results obtained with RRS and from absorption and circular dichroism spectroscopy. The types of interaction and reasons of RRS enhancement are discussed. Linear relationships do exist over a wide range between the intensity of enhanced scattering and the concentrations of either DNA or PMB. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 9.8 ng mL^?1. When PMB is used as a probe to determine DNA, the detection limit (3σ) is in the range from 3.8 to 9.0 ng mL^?1.

Investigation on the interaction between anthracyclines and DNA in the presence of paclitaxel by resonance light scattering technique

We have developed a method to investigate the interaction between DNA-targeted anthracyclines and DNA in the presence of the drug paclitaxel. It is based on resonance light scattering (RLS) and on the finding that anthracyclines when bound to DNA undergo a dramatic enhancement in their RLS intensities, while paclitaxel does not display such an effect. However, the RLS intensities of the anthracyclines-DNA associates are remarkably enhanced again on addition of paclitaxel. UV-visible spectra reveal interactions between paclitaxel and anthracyclines, but no reaction between paclitaxel and DNA. Consequently, paclitaxel, though not DNA-targeted, can improve the DNA-binding capabilities of anthracyclines. Binding constants between anthracyclines and DNA, and improved efficiency of paclitaxel on the DNA-binding capabilities of anthracyclines were calculated. The DNA binding constants of doxorubicin, epirubicin, and mitoxantrone, respectively, are 4.53?×?10⁵?L?mol^?1, 6.05?×?10⁵?L?mol^?1, and 9.47?×?10⁵?L?mol^?1. The improved values in presence of paclitaxel are 78%, 47% and 19%. We also have investigated the effects of drug concentrations and the order of adding the drugs. Displacement studies (using methylene blue as a competitive agent) provided additional information on the mechanisms of the interaction between paclitaxel and anthracyclines.

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50?ng?mL^?1) and the same lower detection limit (0.01?ng?mL^?1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

Detection of DNA hybridization by various spectroscopic methods using the copper tetraphenylporphyrin complex as a probe

We are presenting new and highly sensitive hybridization assays. They are based on various spectroscopic methods including resonance light scattering, circular dichroism, ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy, and relies on the interaction of the Cu(II), Ni(II), Mg(II), Co(II), Cd(II), and Zn(II) complexes, respectively, of tetraphenylporphyrin (TPP) with double-strand DNA (dsDNA) and single strand DNA (ssDNA). The interaction results in amplified resonance light scattering (RLS) signals and enables the detection of hybridization without the need for labeling DNA. The RLS signals are strongest in case of the Cu (II)-TPP complex which therefore was selected as the probe. The technique is simple, robust, accurate, and can be completed in less than one hour.

Sandwich immunoassay for alpha-fetoprotein in human sera using gold nanoparticle and magnetic bead labels along with resonance Rayleigh scattering readout

We describe a sensitive sandwich immunoassay for alpha-fetoprotein (AFP). It is making use of gold nanoparticles (GNPs) and magnetic beads (MBs) as labels, and of resonance Rayleigh scattering for detection. Two antibodies were labeled with GNPs and MBs, respectively, and MB-antigen-GNP complexes were formed in the presence of antigens. The MB labels also serve as solid phase carriers that can be used to magnetically separate the immuno complex. The GNP labels are used as optical probes, and Rayleigh scattering was used to determine the concentration of free GNPs-antibody after separation of the MB-antigen-GNP complexes. The concentration of AFP is related to the intensity of light scattered by free GNPs in the 13.6 pM to 436 pM concentration range, and the limit of detection is 13.6 pM. The method was applied to the determination of AFP in sera of cancer patients, and the results agree well with those obtained by conventional ELISA.

Ultrasensitive electrochemiluminescent immunoassay for morphine using a gold electrode modified with CdS quantum dots, polyamidoamine, and gold nanoparticles

We report on a novel electrochemiluminescent (ECL) immunoassay for the ultrasensitive determination of morphine by making use of a gold electrode which was modified with a nanocomposite film containing self-assembled polyamidoamine (PAMAM) CdS quantum dots and electrodeposited gold nanoparticles (Au-NPs). The highly uniform and well-dispersed quantum dots were capped with PAMAM dendrimers. Due to the synergistic effect of the modified quantum dots and the electrodeposited Au-NPs, the ECL response is dramatically enhanced. Under optimal experimental conditions, the immunoreaction between morphine and anti-morphine antibody resulted in a decrease of the ECL signal because of steric hindrance. The calibration plot is linear in the morphine concentration range from 0.2 to 180 ng?mL^?1, with a detection limit as low as 67 pg?mL^?1. The sensor was successfully applied to the determination of morphine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm^?1 increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8?×?10² to 8?×?10⁶ copies per mL. The limit of detection is 8?×?10² copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection

A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein–bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL^?1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.

A highly sensitive label-free resonance light scattering assay of carcinoembryonic antigen based on immune complexes

As a kind of glycoprotein, carcinoembryonic antigen (CEA) is the important tumor marker for clinical diagnosis of the presence or recurrence of cancer. In this work, a novel label-free resonance light scattering (RLS) spectral CEA assay was developed based on the combination of highly selective immunoreaction and ultrasensitive RLS technique. In Tris–HCl buffer solution (pH 7.5), the specific immunoreaction between CEA antigen and mouse anti-CEA formed immune complexes which had a maximum RLS spectral peak at 389.0 nm, with the existence of physiological saline and polyethylene glycol 20,000 (PEG 20,000). Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI_RLS) was proportional to the concentration of CEA in the range from 0.1 to 60 ng mL⁻¹, with a detection limit (LOD, 3σ) of 0.03 ng mL⁻¹. The characteristics of RLS, the CEA immunocomplex, the immune response, the ratio of CEA antigen and mouse anti-CEA, and the optimum conditions of the immunoreaction have been investigated. The CEA concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) kit. And this method has many satisfying merits including label-free, sensitivity and high selectivity.     

Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL^?1, with a detection limit of 0.3 ng mL^?1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL^?1 of caspase-3 (for n?=?11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n?=?5).

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

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 DNA biosensor based on resonance light scattering using unmodified gold bipyramids

We report on a novel biosensor for determining sequence-specific DNA. It is based on resonance light scattering (RLS) caused by the aggregation of gold bipyramids. These display localized surface plasmon resonance and can be used as a bioprobe. The absorption spectra and the transmission electron micrographs provide visual evidence of the aggregation of the gold bipyramids in the presence of DNA. The RLS intensity of the gold bipyramids increases with the concentration of the target DNA. The method was successfully applied to the determination of a 30-mer single-stranded oligonucleotide and works over the 0.1–10?nM concentration range.

Anodic stripping voltammetry of silver(I) using a carbon paste electrode modified with multi-walled carbon nanotubes

We describe a silver(I)-selective carbon paste electrode modified with multi-walled carbon nanotubes and a silver-chelating Schiff base, and its electrochemical response to Ag(I). Effects of reduction potential and time, accumulation time, pH of the solution and the stripping medium were studied by differential pulse anodic stripping voltammetry and optimized. The findings resulted in a method for the determination of silver over a linear response range (from 0.5 to 235 ng?mL^?1) and with a detection limit as low as 0.08 ng?mL^?1. The sensor displays good repeatability (with the RSD of ±?2.75 % for 7 replicates) and was applied to the determination of Ag(I) in water samples and X-ray photographic films.

Sensitive immunoassay for porcine pseudorabies antibody based on fluorescence signal amplification induced by cation exchange in CdSe nanocrystals

We report on a novel immunoassay for porcine pseudorabies virus (PRV) antibody that is based on fluorescence signal amplification induced by silver(I) ion exchange in CdSe nanocrystals. An antigen-antibody-secondary antibody sandwich structure was first formed from PRV, PRV antibody, and CdSe-labeled rabbit anti-pig antibody. Then, the Cd(II) ions in the CdSe labels were released by a cation exchange reaction with Ag(I). Released Cd(II) was finally quantified using the sensitive fluorescent probe Rhodamine 5 N. Due to this signal amplification, the sensitivity and linear range of the immunoassay were largely improved (compared to the traditional ELISA) in having a limit of detection as low as 1.2 ng?mL^?1 of PRV antibody and a linear range from 2.44 to 312 ng?mL^?1. The successful determination of PRV antibody in pig serum samples is proof for the utility of the method.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Electrochemiluminescence immunoassay at a nanoporous gold leaf electrode and using CdTe quantun dots as labels

An electrochemiluminescence-based immunoassay using quantum dots (QDs) as labels for the carcinoembryonic antigen (CEA) was developed using an electrode modified with leafs of nanoporous gold. CEA was initially immobilized on the electrode via a sandwich immunoreaction, and then CdTe quantum dots capped with thioglycolic acid were used to label the second antibody. The intensity of the ECL of the QDs reflects the quantity of CEA immobilized on the electrode. Thus, in the presence of dithiopersulfate as the coreactant, the ECL serves as the signal for the determination of CEA. The intensity of the electroluminescence (ECL) of the electrode was about 5.5-fold higher than that obtained with a bare gold electrode. The relation between ECL intensity and CEA concentration is linear in the range from 0.05 to 200?ng.mL^-1, and the detection limit is 0.01?ng.mL^-1. The method has the advantages of high sensitivity, good reproducibility and long-term stability, and paves a new avenue for applying quantum dots in ECL-based bioassays.

Interaction between insulin and calf thymus DNA, and quantification of insulin and calf thymus DNA by a resonance Rayleigh scattering method

The interaction of insulin with calf thymus deoxyribonucleic acid (ctDNA) leads to a complex that displays remarkably enhanced resonance Rayleigh scattering (RRS). The complex and its formation were investigated by atomic force microscopy and by absorption, fluorescence and circular dichroism spectroscopies. We show that the Tyr B16, Tyr B26 and Phe B24 amino acids near the active center (Phe B25) were influenced by the interaction, whereas Tyr A14, Tyr A19 and Phe B1 (which are located far away from the active center) were less influenced. The interaction provide a way in the quantitation of both ctDNA and insulin with high sensitivity. When ctDNA is used as a probe to quantify insulin, the detection limit (3σ) is 6.0?ng?mL^-1. If, inversely, insulin is used as a probe to quantify ctDNA, the detection limit (3σ) is 7.2?ng?mL^-1. The analysis of synthetic DNA samples and an insulin infection sample provided satisfactory results.