Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer

A selective kanamycin-binding single-strand DNA (ssDNA) aptamer (TGGGGGTTGAGGCTAAGCCGA) was discovered through in vitro selection using affinity chromatography with kanamycin-immobilized sepharose beads. The selected aptamer has a high affinity for kanamycin and also for kanamycin derivatives such as kanamycin B and tobramycin. The dissociation constants (K_d [kanamycin] = 78.8 nM, K_d [kanamycin B] = 84.5 nM, and K_d [tobramycin] = 103 nM) of the new aptamer were determined by fluorescence intensity analysis using 5′-fluorescein amidite (FAM) modification. Using this aptamer, kanamycin was detected down to 25 nM by the gold nanoparticle-based colorimetric method. Because the designed colorimetric method is simple, easy, and visible to the naked eye, it has advantages that make it useful for the detection of kanamycin. Furthermore, the selected new aptamer has many potential applications as a bioprobe for the detection of kanamycin, kanamycin B, and tobramycin in pharmaceutical preparations and food products.     

Gold nanoparticle-based signal amplification for biosensing

Colloidal gold nanoparticles (AuNPs), with unique properties such as highly resonant particle plasmons, direct visualization of single nanoclusters by scattering of light, catalytic size enhancement by silver deposition, conductivity, and electrochemical properties, are very attractive materials for several applications in biotechnology. Furthermore, as excellent biological tags, AuNPs can be easily conjugated with biomolecules and retain the biochemical activity of the tagged biomolecules, making AuNPs ideal transducers for several biorecognition applications. The goal of this article is to review recent advances of using AuNPs as labels for signal amplification in biosensing applications. We focus on the signal amplification strategies of AuNPs in biosensing/biorecognition, more specifically, on the main optical and electrochemical detection methods that involve AuNP-based biosensing. Particular attention is given to recent advances and trends in sensing applications.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

Gold nanoparticle-based immuno dual probes for targeting proteomics

Immunoprecipitation combined with mass spectrometry (MS) is a promising technique for targeting proteomics in characterizing submicrograms of target protein and interacting proteins in living cells. This method, however, is limited by interference arising from nonspecific binding. We report a novel gold nanoparticle (AuNP)-based immuno probe approach for immunoprecipitation. By cross-linking the antibody Fc domain to protein G covalently modified on AuNPs, the probe was fabricated and characterized to have 60 protein G and 30 immunoglobins per AuNP. We used human immunoglobin against the target and mouse immunoglobin with the same isotype (IgG) to fabricate the target and preclear probe, respectively, and termed it as the dual probe approach. Our results showed that the preclear probe (AuNP-IgG) and the target probe (AuNP-anti-ERα) share a similar panel of nonspecific binders but dramatic different specificity toward the target. Thus, using the dual probe method, we showed major nonspecific binders in the cell lysate could be largely removed without sacrificing the target protein. Compared to the conventional agarose gel-chromatography, the AuNP-based probe exhibited less nonspecific interference and higher recovery yield for ERα. Moreover, the AuNP-based probe is more inert than the agarose gel under harsh conditions and does not induce dissociation of the cross-linked IgG that could interfere with target identification. Using AuNP-based dual probes, ERα was shown to be purified from MCF-7 cells with minimum nonspecific binding. Moreover, the identity and phosphorylation sites on the C-terminus of the purified ERα could be positively confirmed by MS using only 1 mg of cellular protein.     

A gold nanoparticle-based strategy for label-free and colorimetric screening of DNA triplex binders

A label-free colorimetric assay, using non-crosslinking AuNP aggregation, has been developed for the screening of specific triplex DNA binders. The relative binding affinities can be simultaneously determined. Our novel assay is simple in design and fast in operation, avoiding either AuNPs modification or oligonucleotide labeling, and easy to implement for visual detection. This strategy may offer a new approach for developing low cost, sensitive and high-throughput screening platform that is likely to be highly useful in a wide range of applications.     

Biases in the assays of steroids and their binding proteins

Many immunoassays exhibit a bias. In such assays, the results are inaccurate, i.e. they deviate from the true value. The biases are mostly due to interfering compounds originating from the biological material assayed and/or from reagents. Sometimes systematic errors in calculation are also involved. The magnitude of the bias should be determined for every assay method, in order to make possible an assessment of reliability of the method. Biases frequently occur also in the measurements of steroid binding proteins, such as receptors, sex hormone binding globulin, corticosteroid binding globulin, etc. These biases are mostly due to the assumption that the measurements are performed under saturation conditions. These biases can be avoided by conducting the measurements at several concentrations of the ligand and by an appropriate correction for non-specific (low affinity) binding. In the assays of "free" steroids, biases are frequently encountered because of the disturbance of equilibrium in the course of the measurement proper. These biases can be minimized by a careful choice of experimental conditions.     

An azophenolic colorimetric reagent for use in enzyme-linked immunosorbent assays

A colorimetric reagent, 4-(4'-nitro-2'-methylsulfonylphenylazo)phenyl phosphate (NMPP), has been shown to be an effective substrate of alkaline phosphatase. NMPP and p-nitrophenyl phosphate were applied in comparative studies using enzyme immunoassays for the detection of viral antigens and antiviral antibodies. The new substrate exhibited similar, or even higher, sensitivity than p-nitrophenyl phosphate depending on the substrate concentrations used. Positive and negative reactions were easier to define, even without cumbersome equipment. The enzyme reaction was terminated by the uncompetitive inhibitor, theophylline.     

两种新型培养细胞数量检测技术的探索

将SRB比色法和结晶紫比色法与MTT比色法与MTT比色法在96孔板培养HeLa细胞的数量及活力测量精确度和操作方法上进行了对比。试验结果显示：SRB比色法和结晶紫比色法的最佳检测波长均为490nm。最佳染液浓度分别为0.4%和0.25%。SRB比色法的检测精确度略逊于MTT法，结晶紫比色法与MTT法无显著差异。另外，此两种检测方法的操作过程更为简便，所需时间也较短，可在一定范围内取代MTT比色法。     

Comparison of 5 microplate colorimetric assays forin vitro cytotoxicity testing and cell proliferation assays

This paper describes a critical comparative evaluation of 5 miniaturised colorimetric assays applicable to cytotoxicity testing of anti-tumour drugs (and other toxins)in vitro. Each assay shows a different linear range for optical density versus cell number, a different sensitivity to change in cell number and a different minimum detectable cell number; the values of these parameters vary with experimental conditions and with cell line used. All the methods gave good correlation with viable cell number (determined by colony forming efficiency) in toxicity assays after 3 or 4 days of treatment, but they underestimated cell death after 2 days. Toxicity levels for individual chemicals (in a standard 6-day assay) are similar for the different assays, irrespective of the mechanism of action of the chemical being tested. Two of the more recently developed assays (APNaOH and SRB) were found to be very sensitive under the conditions examined.     

A new salting-out technique for colorimetric free fatty acid assays.

A colorimetric assay for measuring long chain (C > 10) free fatty acids in plasma is described. The basic assay procedure of R. J. Schimmel and E. Knobil ((1970) Amer. J. Physiol.218, 1540–1547) was modified in three ways: 1) by not buffering the cobalt-triethanolamine reagent, 2) by replacing potassium sulfate with lithium sulfate, and 3) by making a single reagent of chloroform, heptane, and methanol. In the absence of the buffer system, the soap complex was chemically more stable. A loss of sensitivity incurred by omitting the buffer system was corrected by using a salt solution of 20% (w/w) Na₂SO₄/10% (w/w) Li₂SO₄ instead of saturated Na₂SO₄/K₂SO₄. The number of pipettings was reduced by making a single reagent of chloroform, heptane, and methanol. Because of improved assay stability, large numbers of samples (90) can be processed simultaneously. The method is adequately linear over the range of 100–1250 μequiv/liter. The standard deviation is independent of the mean over the range of 250–1000 μequiv/liter and is about 40 μequiv/liter (n = 15).     

Gold nanoparticle-based surface-enhanced Raman scattering for noninvasive molecular probing of embryonic stem cell differentiation

This study reports the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of mouse embryonic stem (mES) cells, including undifferentiated single cells, embryoid bodies (EBs), and terminally differentiated cardiomyocytes. Gold nanoparticles (GNPs) were successfully delivered into all 3 mES cell differentiation stages without affecting cell viability or proliferation. Transmission electron microscopy (TEM) confirmed the localization of GNPs inside the following cell organelles: mitochondria, secondary lysosome, and endoplasmic reticulum. Using bright- and dark-field imaging, the bright scattering of GNPs and nanoaggregates in all 3 ES cell differentiation stages could be visualized. EB (an early differentiation stage) and terminally differentiated cardiomyocytes both showed SERS peaks specific to metabolic activity in the mitochondria and to protein translation (amide I, amide II, and amide III peaks). These peaks have been rarely identified in undifferentiated single ES cells. Spatiotemporal changes observed in the SERS spectra from terminally differentiated cardiomyocyte tissues revealed local and dynamic molecular interactions as well as transformations during ES cell differentiation.     

Arachidonic acid induced coronary reactions and their inhibition by docosahexaenoic acid

The objective of the present study was to further investigate the influence exerted by docosahexaenoic acid (DHA) on the coronary reactions induced in isolated perfused hearts of rats and guinea pigs by bolus doses of arachidonic acid (AA). As in previous studies, we found that AA produced a coronary constriction followed by a longer lasting dilatation. The present data demonstrate that a 5-min infusion of DHA at 0.17-0.68 microM caused a concentration-dependent inhibition of the AA-induced constriction. The vasodilatation determined by AA was also depressed, but only after about 30 min of a sustained DHA infusion. The precursor of AA, linoleic acid (LA), was also infused for about 30 min, and like DHA it inhibited the coronary reactions induced by AA. LA is not converted into AA by the isolated heart, but like DHA, was probably incorporated into the cells of the coronary vascular compartment. It is known that LA, administered "in vivo" to mammals, is converted into AA and increases the production of eicosanoids, whereas DHA does not follow this metabolic pathway. The incorporation of these essential polyunsaturated fatty acids by the isolated perfused heart would inhibit the cyclooxygenase in the coronary vessel walls, interfering with the generation of vasomotor metabolites from AA. We postulate that the systemic administration of DHA, by inhibiting the synthesis of a constrictor metabolite, could be beneficial in reducing the damage due to microvascular constriction in myocardial ischaemia.     

S-Arachidonoyl-2-thioglycerol synthesis and use for fluorimetric and colorimetric assays of monoacylglycerol lipase

We report the first synthesis of 2-thioglycerol and S-arachidonoyl-2-thioglycerol (the thioester analog of the endocannabinoid 2-arachidonoylglycerol) in an eight or nine step procedure with a yield of ～25% and establish the use of this substrate for maleimide-based fluorescent and dithiobis(2-nitrobenzoic acid)-based colorimetric assays of human recombinant monoacylglycerol (MAG) lipase (hMAGL) and human brain membrane MAG hydrolase activity. Inhibitor structure–activity relationships observed here for hMAGL and 2-ATG correlate well (r² = 0.93, n = 9) with earlier findings for mouse brain MAG hydrolase with non-thiol substrates.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

Computational identification of proteins for selectivity assays

At the stage of optimization of a chemical series the compounds are normally assayed for binding or inhibition on the target protein as well as on several proteins from a selectivity panel. These proteins are normally identified on the basis of sequence homology to the target protein. Experimental selectivity data are also taken into account if available. Cases when a nonhomologous protein has a significant affinity to the compound series are going to be missed if the selectivity panel is identified by homology. Experimental data is usually either unavailable or limited to a small fraction of proteins that should be considered. We have developed a computational method of identification of selectivity panel proteins. It is based on the evaluation of binding site similarity to the target protein using docking scores of target-selected molecular probes. These probes are obtained by docking a large library of drug-like compounds to the target protein followed by selecting a diverse subset from the best virtual binders. Docking scores of these probes to other proteins measure binding site similarity to the target. Because the method does not require prior knowledge of either affinities or structures of inhibitors for the target, it can be applied to any protein with known 3D structure. Validation of the method includes rediscovery of nonhomologous proteins that bind common ligands: estradiol, tamoxifen, and riboflavin. Given 3D structures, the method can effectively discriminate proteins with similar binding sites from random proteins independent of sequence homology.     

Development of a nanoparticle-based surface-modified fluorescence assay for the detection of prion proteins

A nanoparticle-based immunoassay for the detection of recombinant bovine prion protein (PrP) was developed as a step in the development of screening tools for the prevention of the spread of transmissible spongiform encephalopathies. The assay is based on the competitive binding between PrP and a peptide-fluorophore to a nanoparticle-labeled antibody which is specific for a conserved prion sequence. The fluorophore, when bound to the antibody, is subject to surfaced-modified fluorescence, enabling detection of changes in the concentration of bound fluorophore in the presence of prion protein. Important factors considered during the development of the assay were ease of use, robustness, and detection level. The effects of pH and nanoparticle conjugation chemistry on surface-modified fluorescence observed in the assay were explored. Effects of concentrations of antibody and fluorophore on reproducibility and detection limits were examined. At present, the detection limits of the system are approximately equal to the antibody-peptide fluorophore equilibrium dissociation constant, which is near one nanomolar concentration. Improved assay performance could be obtained by optimization of the nanoparticle surface resonance effects. The simplicity of the assay and ease of use may make the type of assay described in this report attractive for screening purposes in the food industry.