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.     

An aptamer based resonance light scattering assay of prostate specific antigen

Prostate specific antigen (PSA) is a valuable tumor marker for prostate cancer screening. In this work, a novel and sensitive resonance light scattering (RLS) spectral assay of PSA was proposed based on PSA aptamer modified gold nanoparticles (AuNPs). The sulfhydryl modified single-strand aptamer could interact with AuNPs, which made the AuNPs stable in high concentration of salt. In pH 7.0 BR buffer solution, the highly selective combination of PSA and AuNPs-labeling aptamer resulted in the aggregation of AuNPs which showed high RLS intensity. Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI(RLS)) was proportional to the concentration of PSA in the range from 0.13 to 110 ng/mL, with a detection limit (LOD, 3σ) of 0.032 ng/mL. This developed RLS assay as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit was successfully applied to the detection of PSA in 15 serum samples, and an excellent correlation of the levels of PSA measured was obtained. This is the first report of the aptamer based RLS assay for PSA and it is also a significant application of instrumental analysis technique.     

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.     

绿色木霉菌合成金纳米颗粒的可能性及影响因素

【背景】金纳米颗粒(AuNPs)凭借其稳定性、抗氧化性能和生物相容性在许多领域有广泛应用。目前关于微生物合成金纳米颗粒的研究较少。【目的】对微生物合成金纳米颗粒的可能性以及影响因素进行探究,有利于揭示具体的合成机制,发现AuNPs的特性以及合成位置与菌丝和影响因素的关系。【方法】以绿色木霉菌(Trichoderma viride)菌株(GIM3.141)为菌种资源,通过目视检测法、紫外可见分光光度计、X射线衍射和透射电镜等手段分析合成AuNPs的特征。探讨细胞内生物合成金纳米颗粒(AuNPs)的可能性,研究生物量、初始金离子浓度、溶液pH等因素对细胞内合成AuNPs的影响。【结果】X射线衍射分析表明AuNPs以金纳米晶体形态存在。透射电镜分析表明AuNPs主要位于细胞壁膜间隙,一小部分附着在细胞壁上。紫外可见分光光度计分析表明,金纳米颗粒粒径随着生物量添加量和溶液pH的升高而变小,随着初始金离子浓度的升高而变大。【结论】非致病性真菌绿色木霉菌可以在细胞内合成AuNPs,其中包括伪球形、三角形、四边形和六边形等多种形状,粒径范围从几纳米到三百纳米,为大规模、低成本、无污染地生物合成纳米颗粒工艺提供了菌种资源。     

Environmentally benign rapid biosynthesis of extracellular gold nanoparticles using Aspergillus flavus and their cytotoxic and catalytic activities

In this study, the rapid biosynthesis of gold nanoparticles (AuNPs) by Aspergillus flavus culture supernatant was achieved by reducing 1 mM of chloroauric acid (HAuCl₄) within 2 min at pH 7 and 30 °C. The biosynthesized nanoparticles exhibited maximum absorbance at 545 nm in UVvis spectroscopy. Transmission electron microscopy exhibited that AuNPs tend to take nearly spherical shapes with an average size of 12 nm. Fourier transform infrared analysis indicated that carboxyl, amine, and hydroxyl groups may participate in the biosynthesis and stabilization of AuNPs. Its zeta potential was found to be -33.01 mV. Energy dispersive X-rays showed a strong and typical beak of gold nanocrystallites with 80.84 % of analyzed sample. X-Ray diffraction spectrum displayed Bragg reflections identical to the gold nanocrystals. The results confirmed that biosynthesized AuNPs are a potent anticancer agent against A549, HepG2 and MCF7 cell lines with IC₅₀ value 53.5, 60.7 and 100 μg/mL, respectively. Crystal violet assay confirmed the cytopathic effects of AuNPs on HepG2 and A549 cell lines. Annexin-V FITC assay and cell cycle confirmed the apoptotic effect and cell cycle arrest in G2/M phase, respectively for A549 cell line. Moreover, the results showed a degradation efficiency of AuNPs to 4-nitrophenol within 16 min.     

Optical sensing of biomedically important polyionic drugs using nano-sized gold particles

A simple optical method for the sensing of biomedically important polyionic drugs, protamine and heparin based on the reversible aggregation and de-aggregation of gold nanoparticles (AuNPs) is described. The polycationic protamine induces the aggregation of negatively charged citrate-stabilized AuNPs, resulting in a shift in the surface plasmon (SP) band and a consequent color change of the AuNPs from red to blue. Addition of polyanionic heparin dissipates the aggregated AuNPs due to its strong affinity to protamine and the blue color changes to the native color. The color change was monitored using UV-vis spectrophotometry. The aggregation and de-aggregation was confirmed by transmission electron microscopic (TEM) measurements. The degree of aggregation and de-aggregation is proportional to the concentration of added protamine and heparin, allowing their quantitative detection. The change in the absorbance and SP band position has been used to monitor the concentration of protamine and heparin. This optical method can quantify protamine and heparin as low as 0.1 microg/ml and 0.6 microg/ml, respectively and the calibration is linear for a wide range of concentration.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

Selective recognition of homocysteine and cysteine based on new ruthenium(II) complexes

A series of the new ruthenium(II) complexes with different number of aldehyde groups have been synthesized and characterized for the simple and selective sensing of homocysteine (Hcy) and cysteine (Cys). The reaction of these ruthenium(II) complexes with Hcy and Cys afforded thiazinane or thiazolidine derivatives which resulted in the obvious changes in the UV-visible spectra and strong enhancement of the luminescence intensity of the system. The luminescence enhancement of [Ru(dmb)₂(L2)]²⁺ (dmb: 4,4′-dimethyl-2,2′-bipyridine) showed a good linearity in the concentration of 4.2-350 μM and 6-385 μM with the detection limits of 0.3 μM and 1 μM for Hcy and Cys, respectively. The absorption and emission bands from metal-to-ligand charge transfer transition in the visible region and the large Stokes shift of the ruthenium(II) complex chromophore made it suitable for biological applications.     

Sensitive detection of estradiol based on ligand binding domain of estrogen receptor and gold nanoparticles

With increasing concerns of estrogenic effects of endocrine disrupting compounds, the development of simple detection assay for these compounds is an ongoing need. Herein, a simple, rapid, and highly sensitive assay for estradiol (E2) detection was developed using the ligand binding domain of estrogen receptor α (LBD-ERα), the receptor interacting domain of steroid receptor co-activator 1 (RID-SRC1), and gold nanoparticles (AuNPs). The colloidal AuNPs could be stabilized against a salt-induced aggregation by adding LBD-ERα protein. However, with the presence of E2, the specific binding of LBD-ERα protein and E2 led to a salt-induced aggregation of AuNPs as seeing from a color change from red to blue. This developed assay exhibited a high sensitivity for E2 detection with the limit of detection (LOD) of 2.62 × 10⁻¹⁴ M. When the RID-SRC1 protein was included, the detection sensitivity was increased, which the LOD for E2 was at 1.20 × 10⁻¹⁵ M. This assay was specific for a detection of E2 but not progesterone, the negative control ligand. Results of this work clearly showed the efficiency of developed assay for E2 detection, which possibly further developed for an onsite monitoring of E2.     

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.     

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.     

Plasmonic Spectral Detection of Carcinoembryonic Antigen by Preventing the Direct Binding of Rhodamine 6G with Au Nanoparticles

Carcinoembryonic antigen (CEA) was used as a separator to prevent the Rhodamine 6G (R6G)-induced aggregation of colloidal gold nanoparticles. The destroyed aggregation has been monitored by measuring the absorption and resonance light scattering peaks corresponding to the longitudinal surface plasmon resonance (SPR) of the chain-like aggregated gold nanoparticles (AuNPs). It was found that the pre-adding of CEA with different concentrations to the gold colloids before mixing them with R6G could lead to the longitudinal SPR peak decrease and blue shift. By analysing the intensity changing and wavelength shifting of the absorption spectra, CEA could be detected in a linear range from 0.2 to 4 ng/mL, and the limit of detection reaches to 0.1 ng/mL. The sensitivity of the CEA concentration dependent shifting and quenching of the plasmonic absorption and scattering corresponding to the AuNPs aggregation presents a well potential application of biologic spectral sensing.     

Oligonucleotide-stabilized fluorescent silver nanoclusters for sensitive detection of biothiols in biological fluids

In this work, we reported a simple and sensitive method to detect biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), using fluorescent silver nanoclusters (Ag NCs) stabilized by single-stranded DNA (DNA-Ag NCs) as probes. The photoluminescence intensity of DNA-Ag NCs was found to be quenched effectively with the increase of biothiols concentration due to the formed nonfluorescent coordination complex between DNA-Ag NCs and biothiols, resulting in the shift-to-red of emission wavelength. But the fluorescence of DNA-Ag NCs was not changed in the presence of other amino acids at 10-fold higher concentration. Satisfactory detection limits and linear relationships of Cys, GSH and Hcy were obtained, respectively. The resulted plots exhibited good linear relationships in the range from 8.0×10(-9) to 1.0×10(-7) mol L(-1) (R(2)=0.984) for Cys, 8.0×10(-9) to 1.0×10(-7) mol L(-1) (R(2)=0.983) for GSH, and 2.0×10(-6) to 6.0×10(-7) mol L(-1) (R(2)=0.999) for Hcy, respectively; the detection limits of Cys, GSH and Hcy were 4.0 nmol L(-1), 4.0 nmol L(-1), and 0.2 μmol L(-1), respectively. The method was successfully used for the detection of biothiols in human plasma samples.     

Enzyme-nanoparticle conjugates at oil-water interface for amplification of electrochemical immunosensing

A novel cholesterol biosensor was prepared based on gold nanoparticles-catalyzed luminol electrogenerated chemiluminescence (ECL). Firstly, l-cysteine-reduced graphene oxide composites were modified on the surface of a glassy carbon electrode. Then, gold nanoparticles (AuNPs) were self-assembled on it. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the surface of AuNPs to construct a cholesterol biosensor. The stepwise fabrication processes were characterized with cyclic voltammetry and atomic force microscopy. The ECL behaviors of the biosensor were also investigated. It was found that AuNPs not only provided larger surface area for higher ChOx loading but also formed the nano-structured interface on the electrode surface to improve the analytical performance of the ECL biosensor for cholesterol. Besides, based on the efficient catalytic ability of AuNPs to luminol ECL, the response of the biosensor to cholesterol was linear range from 3.3 μM to 1.0 mM with a detection limit of 1.1 μM (S/N=3). In addition, the prepared ECL biosensor exhibited satisfying reproducibility, stability and selectivity. Taking into account the advantages of ECL, we confidently expect that ECL would have potential applications in biotechnology and clinical diagnosis.     

Colorimetric detection of bisphenol A based on unmodified aptamer and cationic polymer aggregated gold nanoparticles

In this study, a colorimetric method was exploited to detect bisphenol A (BPA) based on BPA-specific aptamer and cationic polymer-induced aggregation of gold nanoparticles (AuNPs). The principle of this assay is very classical. The aggregation of AuNPs was induced by the concentration of cationic polymer, which is controlled by specific recognition of aptamer with BPA and the reaction of aptamer and cationic polymer forming “duplex” structure. This method enables colorimetric detection of BPA with selectivity and a detection limit of 1.50 nM. In addition, this colorimetric method was successfully used to determine spiked BPA in tap water and river water samples.     

Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: a glucose biosensor application

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.     

Ratiometric fluorescence detection of superoxide anion based on AuNPs‐BSA@Tb/GMP nanoscale coordination polymers

A novel ratiometric fluorescence nanosensor for superoxide anion (O₂^??) detection was designed with gold nanoparticles‐bovine serum albumin (AuNPs‐BSA)@terbium/guanosine monophosphate disodium (Tb/GMP) nanoscale coordination polymers (NCPs) (AuNPs‐BSA@Tb/GMP NCPs). The abundant hydroxyl and amino groups of AuNPs‐BSA acted as binding points for the self‐assembly of Tb³⁺ and GMP to form core‐shell AuNPs‐BSA@Tb/GMP NCP nanosensors. The obtained probe exhibited the characteristic fluorescence emission of both AuNPs‐BSA and Tb/GMP NCPs. The AuNPs‐BSA not only acted as a template to accelerate the growth of Tb/GMP NCPs, but also could be used as the reference fluorescence for the detection of O₂^??. The resulting AuNPs‐BSA@Tb/GMP NCP ratiometric fluorescence nanosensor for the detection of O₂^?? demonstrated high sensitivity and selectivity with a wide linear response range (14 nM–10 μM) and a low detection limit (4.7 nM).     

Folic acid incorporated nitrogen-doped carbon dots as a turn-on fluorescence probe for homocysteine detection

This study describes the development of a low-cost fluorescence assay for detecting homocysteine (Hcy) without the interference of cysteine and glutathione using carbon quantum dots. Herein nitrogen-doped carbon quantum dots (NCDs) were synthesized from citric acid as the carbon source and urea as the dopant using a one-pot microwave-assisted method. The obtained NCDs were incorporated with folic acid (FA) by the direct ex situ addition method and were used as a fluorescence probe to detect Hcy. The probe exhibited a fluorescence turn-on response with increased Hcy concentration up to 50 μM with a limit of detection of 2.276 μM. The point of care detection of Hcy using the probe was also tested with a paper-based assay strip.     

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.