A novel label-free upconversion fluorescence resonance energy transfer-nanosensor for ultrasensitive detection of protamine and heparin

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs–AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2 μg/ml and 0.002 to 2.0 μg/ml with low detection limits of 6.7 and 0.7 ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.     

Colorimetric detection of melamine in milk by citrate-stabilized gold nanoparticles

Here, we report a simple and sensitive colorimetric method for detection of melamine in milk using gold nanoparticles (AuNPs). AuNPs of 21-nm size were synthesized by the citrate reduction method. The method is based on the principle that the melamine causes the aggregation of AuNPs and, hence, the wine red color of AuNPs changes to blue or purple. This change in color can be visualized with the naked eye or an ultraviolet–visible (UV–Vis) spectrometer. Under optimized conditions, AuNPs are highly specific for melamine and can detect melamine down to a concentration of 0.05 mg L⁻¹.     

Sensitive colorimetric visualization of dihydronicotinamide adenine dinucleotide based on anti-aggregation of gold nanoparticles via boronic acid-diol binding

A facile, highly sensitive colorimetric strategy for dihydronicotinamide adenine dinucleotide (NADH) detection is proposed based on anti-aggregation of gold nanoparticles (AuNPs) via boronic acid-diol binding chemistry. The aggregation agent, 4-mercaptophenylboronic acid (MPBA), has specific affinity for AuNPs through Au-S interaction, leading to the aggregation of AuNPs by self-dehydration condensation at a certain concentration, which is responsible for a visible color change of AuNPs from wine red to blue. With the addition of NADH, MPBA would prefer reacting with NADH to form stable borate ester via boronic acid-diol binding dependent on the pH and solvent, revealing an obvious color change from blue to red with increasing the concentration of NADH. The anti-aggregation effect of NADH on AuNPs was seen by the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for NADH is from 8.0 × 10(-9)M to 8.0 × 10(-6)M, with a low detection limit of 2.0 nM. The as-established colorimetric strategy opened a new avenue for NADH determination.     

Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTA's aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTA's aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.     

Label-free colorimetric sensor for ultrasensitive detection of heparin based on color quenching of gold nanorods by graphene oxide

A novel label-free colorimetric strategy was developed for ultrasensitive detection of heparin by using the super color quenching capacity of graphene oxide (GO). Hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorods (AuNRs) could easily self-assembly onto the surface of GO through electrostatic interaction, resulting in decrease of the surface plasmon resonance (SPR) absorption and consequent color quenching change of the AuNRs from deep to light. Polycationic protamine was used as a medium for disturbing the electrostatic interaction between AuNRs and GO. The AuNRs were prevented from being adsorbed onto the surface of GO because of the stronger interaction between protamine and GO, showing a native color of the AuNRs. On the contrary, in the presence of heparin, which was more easily to combine with protamine, the AuNRs could self-assembly onto the surface of GO, resulting in the native color disappearing of AuNRs. As the concentration of heparin increased, the color of AuNRs would gradually fade until almost colorless. The amounts of self-assembly AuNRs were proportional to the concentration of heparin, and thereby the changes in the SPR absorption and color had been used to monitor heparin levels. Under optimized conditions, good linearity was obtained in a range of 0.02-0.28 μg/mL (R=0.9957), and a limit of detection was 5 ng/mL. The simultaneous possession of high sensitivity and selectivity, simplicity, rapidity, and visualization enabled this sensor to be potentially applicable for ultrasensitive and rapid on-site detection toward trace heparin.     

Colorimetric sensing of clenbuterol using gold nanoparticles in the presence of melamine

A highly sensitive method for the detection of trace amount of clenbuterol based on gold nanoparticles (AuNPs) in the presence of melamine was described in this paper. Hydrogen-bonding interaction between clenbuterol and melamine resulted in the aggregation of AuNPs and a consequent color change of AuNPs from wine red to blue. The concentration of clenbuterol could be determined with naked eye or a UV-vis spectrometer. Results showed that the absorption ratio (A(670)/A(520)) was liner with the logarithm of clenbuterol concentration in the range of 2.8×10(-10) to 2.8×10(-7)M and 2.8×10(-7) to 1.4×10(-6)M with linear coefficients of 0.996 and 0.993, respectively. The detection limit was 2.8×10(-11)M (S/N=3), which was much lower than most existing methods. The coexisting substances including dl-epinephrine, phenylalamine, tryptohan, alamine, uric acid, glycine, glycerol, glucose, MgCl(2), CaCl(2) and NaCl did not affect the determination of clenbuterol. The proposed method could be successfully applied to the determination of clenbuterol in human urine.     

Simple colorimetric sensing of trace bleomycin using unmodified gold nanoparticles

A simple colorimetric sensing platform for trace bleomycin (BLM) was proposed with the unmodified gold nanoparticles (AuNPs) as the sensing element. BLM has multiple N-donor functionality and exhibited strong coordination effect on AuNPs, which made it possible for the occurrence of ligand exchange of BLM with the weakly surface-bound citrate ions on AuNPs. Meanwhile, the positively charged BLM molecules further neutralized the surface charge, leading to increased van der Waals attractive force among AuNPs for rapid aggregation. This was reflected by the obvious color change from wine red to blue and rapid aggregation kinetics within 7.5 min. The BLM sensing based on unmodified AuNPs can be seen with the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for BLM was from 2 to 150 nM. The as-established colorimetric strategy opened a new avenue for trace BLM determination.     

Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase,a Biomarker for Malaria,Using the Specific DNA Aptamer

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.     

A simple, label-free AuNPs-based colorimetric ultrasensitive detection of nerve agents and highly toxic organophosphate pesticide

Here, a simple label-free colorimetric sensing method for organophosphate (OP) nerve agents and pesticide based on catalytic reaction of acetylcholine esterase (AChE) and the aggregation of lipoic acid (LA) capped AuNPs has been established, which is highly sensitive with a limit of detection (LOD) lowered to pM level. In this method, only the AChE hydrolysis product of acetylthiocholine (ATCh), i.e., cationic thiocholine (TCh) can induce the aggregation of LA capped AuNPs along with a distinct color change from red to steel-blue. When OPs as enzyme inhibitors exist, the generation of TCh can be suppressed and the color change of LA capped AuNPs is gradually diminished according to different concentrations of OPs. The feasibility of this method has been demonstrated by sensitive measurement of OP nerve agents and pesticide in a spiked fruit sample with reliable results. This distinct and rapid colorimetric response enables us to readily probe OPs without more technical demand.     

Aqueous zymography screening of matrix metalloproteinase activity and inhibition based on colorimetric gold nanoparticles

An optical gold nanoparticles (AuNPs)-based method was fabricated for the rapid detection of matrix metalloproteinase (MMP) activity and screening potential MMP inhibitors without sophisticated instruments. The diagnosis platform was composed of AuNPs, particular MMP substrates and 6-mercapto-1-hexanol (MCH). The functionalized AuNPs were subjected to specific MMP digestion, and the MMP found the substrate on AuNPs, such that the AuNPs lost shelter and MCH increased the attraction force between AuNPs. Consequently, AuNPs aggregation and a color change from red to purple with increasing MMP concentration were observed. The surface plasmon resonance (SPR) of the formed AuNPs allowed for the quantitative detection of MMP activity. A sensitive linear correlation existed between the absorbance and the activity of the MMPs, which ranged from 10 ng/mL to 700 ng/mL in NTTC buffer and plasma samples. The proposed colorimetric method could be accomplished in a homogeneous solution with one-step operation in 30 min and has been successfully applied to the determination of particular MMP activity in plasma samples, in which the results are consistent with substrate zymography. This technology may become a simple platform for parallel screening a number of inhibitors and offer an alternative method to studying the efficiency of inhibitors for suppressing MMP activity. The absorbance ratio at 625 nm and 525 nm (A(625)/A(525)) confirmed the efficiency of the inhibitors as observed in substrate zymography. The IC(50) of ONO-4817 and galardin for MMP-1, MMP-2 and MMP-7 determined by the proposed colorimetric method was similar to the results of substrate zymography.     

Titrimetric method for determination of O-desulfated heparin in physiological samples using protamine-sensitive membrane electrode as endpoint detector

O-Desulfated heparin (ODSH) is a promising new anti-inflammatory agent for the prevention of reperfusion injury following myocardial infarction or stroke. This partially desulfated heparin derivative has less anticoagulant activity than unfractionated heparin but retains the inherent anti-inflammatory properties of heparin. Thus, ODSH could be administered at the high doses needed to achieve desired anti-inflammatory function without risk of hemorrhage. However, given the very low anticoagulant activity of this species, traditional methods for heparin determination in clinical samples might not be well suited for ODSH measurements. In this article, a novel titrimetric method for detection of ODSH in buffer and plasma is described using a protamine-sensitive polymer membrane electrode as the detector. Titrations of ODSH with the heparin antagonist protamine yield sharp endpoints with sensitivity to ODSH in the micrograms per milliliter range for plasma samples. The stoichiometry for protamine interaction with ODSH is determined to average 1.39 microg protamine/microg ODSH in plasma. This technology is further applied to a toxicokinetic study of ODSH in an animal model, demonstrating the ability to detect the changes in ODSH concentrations in biological samples.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Label-free colorimetric and quantitative detection of cancer marker protein using noncrosslinking aggregation of Au/Ag nanoparticles induced by target-specific peptide probe

Assays for non-enzyme protein based on peptide-protein interaction are few due to the fact that most of peptide-protein bindings do not produce easily measurable output signals. Here we report a homogenous assay for colorimetric and quantitative detection of a cancer marker and promising antitumor target, cyclin A(2), using noncrosslinking aggregation of unmodified AuNPs/AgNPs by utilizing the difference of coagulating ability of a cationic peptide probe (P1) and its binding form toward naked AuNPs/AgNPs. In the absence of cyclin A(2), P1 coagulates particles immediately, whereas cyclin A(2) binding prevents the interaction of P1 with metal particles surface, significantly reducing the magnitude of aggregation. The extent of aggregation is dependent on the concentration of the target protein cyclin A(2) and the difference in color can readily be distinguished by spectrometer and naked eyes. The assay is sensitive and selective. Cyclin A(2) assay using AuNPs as colorimetric indicator is more easily monitored by naked eyes owing to the distinct color change, and 40 nM cyclin A(2) can be detected without the aid of any instruments. Using inexpensive desktop spectrometer, cyclin A(2) assay using AgNPs as colorimetric indicator can detect as low as 30 nM cyclin A(2), which is 20 fold lower than that of cyclin A(2) assay using terbium-chelating peptide as the probe reported recently (Pazos et al., 2008, 130, 9652-9653). This strategy will shed light on developing of unlabeled peptide-based protein biosensors.     

Differential mechanisms for structural and functional alterations of trypsin by heparin, evidence for a specific, radical-generating mechanism at low heparin concentrations

The oxidative mechanism whereby heparin may interact with various proteins was investigated in detail in this work by addressing the role of doses of heparin on the nature and effects of its binding to bovine trypsin, taken as reference protein. Unfractionated heparin was used at concentrations ranging from 6 to 400 microg/ml with a fixed trypsin concentration (250 microg/ml). At concentrations of up to 60 microg/ml, equivalent to trypsin/heparin molar ratios of between 30 and 3, increasing inhibition of amidolytic activity and radical-dependent peptide bond cleavage of the enzyme was observed, with the appearance in the electrophoretic pattern of new bands of trypsin fragments to which heparin was demonstrated to be bound specifically. Structural modifications were also revealed by increases in fluorescence emission spectra. On the whole, however, the alterations induced by these heparin concentrations only involved a limited number of trypsin molecules. At concentrations from 120 to 400 microg/ml (equivalent trypsin/heparin molar ratios of 1.5-0.46), heparin binding to trypsin appeared to cause more profound and generalized alterations of enzyme structure and function, with dose-dependent quenching of fluorescence emission and almost complete loss of amidolytic activity, although evidence of radical production was lacking. Collectively, the results stress the crucial role of heparin dose on both the nature and effects of its binding to trypsin. The change in heparin effects which reflects distinct underlying molecular mechanisms occurs dramatically at a critical concentration threshold. While a specific, radical-generating mechanism operates at low concentrations, less specific ionic linkages, apparently independent of radical production, best explain the effects of high heparin concentrations.     

A highly sensitive resonance Rayleigh scattering and colorimetric assay for the recognition of propranolol in β‐adrenergic blocker

In this work, a highly sensitive, citrate anion‐capped gold nanoparticles (AuNPs)‐based assay for the determination of propranolol in real samples with resonance Rayleigh scattering (RRS) and colorimetry was developed. When AuNPs were prepared by the sodium citrate reduction method, citrate anions self‐assembled on the surface of AuNPs to form supramolecular complex anions. In BR 4.6 buffer solution, propranolol was positively charged and could bind with AuNPs to form larger aggregates through electrostatic force and hydrophobic effects. This results in remarkable enhancement of the RRS intensity and a color change in the AuNPs solution from red to blue via purple. Thus, a highly sensitive RRS and colorimetric assay the for detection of propranolol was developed with a linear range of 0.2–5.2 and 8–112 ng/ml, respectively. In addition, no difference was seen when comparing R‐propranolol with S‐propranolol, therefore, this method could not be used in the recognition of chiral propranolol. However, upon addition of other β‐adrenergic blockers, no phenomenon like that seen with propranolol was observed, meaning that this method can be used for determining the presence of propranolol in a mixture β‐adrenergic blockers. Finally, the optimum conditions, factors influencing the reaction, its mechanism and the reasons for enhancement of the RRS were discussed.     

An improved micromethod for tyrosine estimation

A modified and improved micromethod for tyrosine determination has been developed. The method is sensitive, economic and applicable for estimation of tyrosine released in enzymatic reactions and in tissue. A range of Folin-Ciocalteu (FC) reagent was used to optimize the conditions for the development of blue color. Thus in 1.5 ml of the assay system, the suitably diluted FC reagent at the final concentration of 0.2 N gave a rapid optimum color development with an absorption maximum at 750 nm. Color development showed a linear relationship in the range of 2 to 16 microg tyrosine for a described assay system under optimized conditions. Thus, the method is 3-fold more sensitive in terms of its estimation range than a conventional method. The blue color formed was stable up to 24 h. The applicability of the method for tyrosine determination in the assay of lysosomal cathepsin D and in tissue was checked by comparison to the conventional procedure. Under both systems the results obtained by the micromethod were identical to those obtained by the conventional method. In general the method that produces quantitatively a blue color, not only is rapid and economical in terms of chemical usage but also has application for routine biochemical analysis.     

Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay

We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.     

A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays

A simplified method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized. Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid per milliliter of 1.0 N H2SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development showed a linear relationship up to 10 microg Pi concentration. Thus the method has a 2.5x higher range of Pi estimation than that of the Bartlett method. The molar extinction coefficient at 820 nm was higher than that obtained in the Bartlett procedure. Also the molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate as in the case of Na+,K+ ATPase was at the minimum. The applicability of the method for assay of microsomal Na+,K+ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3-4 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid economical, and convenient but also has wide applicability.     

A less toxic heparin antagonist--low molecular weight protamine

A new thirteen amino acid peptide, named low molecular weight protamine (LMWP), was obtained through the enzymatic digestion of native protamine. Both in vitro and in vivo results showed that LMWP fully maintained the heparin neutralization function of protamine but had much lower immunogenicity and antigenicity. Unlike protamine, neither LMWP nor LMWP/heparin complexes caused significant blood platelet aggregation in rats. These results suggest that LMWP can be used as a substitute for protamine for developing a new generation of nontoxic heparin antagonists.     

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.