Turn‐On Fluorescence Sensor Based on Single‐Walled‐Carbon‐Nanohorn–Peptide Complex for the Detection of Thrombin

Proteases play a central role in several widespread diseases. Thus, there is a great need for the fast and sensitive detection of various proteolytic enzymes. Herein, we have developed a carbon nanotube (CNT)‐based protease biosensing platform that uses peptides as a fluorescence probe for the first time. Single‐walled carbon nanohorns (SWCNHs) and thrombin were used to demonstrate this detection strategy. SWCNHs can adsorb a fluorescein‐based dye (FAM)‐labeled peptide (FAM‐pep) and quench the fluorescence of FAM. In contrast, thrombin can cleave FAM‐pep on SWCNHs and recover the fluorescence of FAM, which allows the sensitive detection of thrombin. This biosensor has a high sensitivity and selectivity toward thrombin, with a detection limit of 100 pM .     

Acid cleavable surface enhanced raman tagging for protein detection

Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ～5 nM in terms of the BSA concentration and ～1.5 nM in ACST content. The dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ～10 nM to ～100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.     

Fluorescence correlation spectroscopy of gold nanoparticles,and its application to an aptamer-based homogeneous thrombin assay

We have studied the fluorescence properties and diffusion behaviors of gold nanoparticles (GNPs) in solution by using fluorescence correlation spectroscopy (FCS) at single molecule level. The GNPs display a high photo-saturation feature. Under illumination with strong laser light, they display higher brightness per particle (BPP) despite their low quantum yields. Based on the unique fluorescence properties and diffusion behaviors of GNPs, we have developed a sensitive and homogenous thrombin assay. It is based on a sandwich strategy and is making use of GNPs to which two different aptamers are conjugated. When the differently aptamer-labeled GNPs are mixed with solutions containing thrombin, the affinity reaction causes the GNPs to form dimers or oligomers. This leads to an increase in the diffusion time of the GNPs in the detection volume that is seen in FCS. The FCS method enables sensitive detection of the change in the characteristic diffusion time of the GNPs before and after the affinity reaction. Quantitative analysis of thrombin is based on the measurement of the change in the diffusion time. Under optimal conditions, the calibration plot is linear in the 0.5 nM to 110 nM thrombin concentration range, and the detection limit is 0.5 nM. The method was successfully applied to the direct determination of thrombin in human plasma.

Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13-4 nM and 0.56-12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.     

Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence

Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter 12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated before Au-NPs are included in any qPCR assay. Figure Raw amplification profiles in the presence and absence of gold nanoparticles     

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.     

Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection

In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin 15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin 29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity. Results showed that thrombin was retained at the aptamer-modified glass surface while nonspecific proteins were removed by rinsing with buffer solution. This approach successfully showed the suitability of aptamers as low molecular weight receptors on glass slides for sensitive and specific protein detection.     

Gold nanorods-based FRET assay for sensitive detection of Pb2+ using 8-17DNAzyme

In this paper, we have reported a sensitive assay for fluorescence "turn-on" detection of Pb(2+) in aqueous solutions based on FRET between gold nanorods (GNRs) and the FAM-labeled substrate strand of 8-17DNAzyme. The fluorescence of the FAM-labeled substrate strand is quenched when 8-17DNAzyme is adsorbed on GNRs surface through electrostatic interaction. In the presence of lead ions, the fluorescence is restored due to the decrease of FRET efficiency caused by the specific cleavage of the FAM-labeled substrate strand by the enzyme, which weakens the electrostatic interaction between the GNRs and short FAM-labeled DNA fragment. The interference of eleven common metal ions has been tested, indicating that Pb(2+) can be selectively detected. This method exhibits a high sensitivity for Pb(2+) with a detection limit of 61.8 pM and a linear range from 0.1 nM to 100 nM. It is a simple, sensitive, and selective method for Pb(2+) detection. Moreover, this sensing system obtained satisfying results for Pb(2+) detection in tap water samples.     

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform.     

Sensitive Electrochemical Aptasensor for Thrombin Detection Based on Graphene Served as Platform and Graphene Oxide as Enhancer

A sensitive electrochemical aptasensor was developed with conductive graphene served as platform and inert graphene oxide (GO) as enhancer. An electrodeposited nano-Au layer was firstly formed on conductive graphene modified glass carbon electrode surface for further immobilizing of electrochemical redox probe hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was formed for immobilizing of thrombin aptamer (TBA). In the presence of thrombin, the TBA on the electrode surface could bind with thrombin, which made a barrier for electrons and inhibited the electro-transfer, resulting in the decreased electrochemical signals of NiHCFNPs. Owing to the non-conductivity property of graphene oxide, further decreased electrochemical signals of NiHCFNPs could be obtained via the sandwich reaction with GO-labeled TBA. According to the signal changes before the thrombin recognition and after sandwich reaction, trace detection of thrombin could be achieved. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range from 0.005 nM to 50 nM with a detection limit of 1 pM.     

Determination of Lysozyme by Graphene Oxide–Polyethylene Glycol-Based Fluorescence Resonance Energy Transfer

The surface of graphene oxide was modified by bis(3-aminopropyl)-terminated polyethylene glycol to produce a composite graphene–polyethylene glycol. The graphene oxide/polyethylene glycol maximum absorption peak in the ultraviolet–visible spectrum was redshifted, and transmission electron microscope images showed that graphene oxide was cleaved into small nanosheets to form graphene oxide/polyethylene glycol. The dispersibility of graphene oxide/polyethylene glycol in physiological solution was higher than for graphene oxide. The optimum composite of graphene oxide/polyethylene glycol was used as a quencher in a fluorescence resonance energy transfer aptasensor for the determination of lysozyme detection. The results showed that graphene oxide/polyethylene glycol rapidly and efficiently quenched the fluorescence of the dye-labeled aptamer. The fluorescence was recovered by adding lysozyme to the system. The aptamer fluorescence intensity exhibited a strong linear dependence on the lysozyme concentration from 50 to 300?nM, and the lysozyme detection limit was approximately 11?nM. This method was used for the determination of lysozyme in egg whites, demonstrating that this approach is a promising alternative for the determination of lysozyme.     

Label-free aptamer biosensor for selective detection of thrombin

We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd²⁺ on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results.     

Simple optical determination of silver ion in aqueous solutions using benzo crown-ether modified gold nanoparticles

We describe a method for the modification of gold nanoparticles (Au-NPs) with benzo-15-crown-5 that led to the development of a colorimetric assay for Ag(I) ion. The brown color of a solution of the modified Au-NPs turns to purple on addition of Ag(I) ion. The ratio of the UV–vis absorption at 600 nm and 525 nm is proportional to the concentration of Ag(I) ions in the range from 20 to 950 nM, and the detection limit is 12.5 nM. Other metal ions do not interfere if present in up to millimolar concentrations. The method enables a rapid determination of Ag(I) in lake and drinking water and is amenable to bare-eye readout.

Dual-luminophore-labeled gold nanoparticles with completely resolved emission for the simultaneous imaging of MMP-2 and MMP-7 in living cells under single wavelength excitation

Although considerable effort has been devoted to the design of various nanoprobes for the fluorescent detection of multiple biomarkers in a single assay, they often suffer from emission-overlapping, owing to small Stokes shifts and wide emission spectra, which results in cross-talk and inaccurate quantification. Herein, we report the design and synthesis of a new nanoprobe for multienzyme detection with completely resolved emission peaks under single-wavelength excitation. The probe was assembled by attaching a cleavable peptide spacer, which was comprised from a matrix metalloproteinase-2 (MMP-2) substrate and a MMP-7 substrate, onto the surface of gold nanoparticles (AuNPs) through cysteine residues. A lanthanide complex, BCTOT-Eu(III) (BCTOT=1,10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone), and 7-amino-4-methylcoumarin (AMC) were attached to the N terminus and the C terminus of the peptide, respectively. In the presence of one or both targeting enzymes, the substrate was cleaved and fluorescence resonance energy transfer (FRET) between the dyes and AuNPs was prohibited, thereby resulting in the dramatic fluorescence emission of dyes. Importantly, there was no emission cross-talk between the two dyes, thereby ensuring accurate detection of each enzyme. Based on this, the simultaneous fluorescence image of MMP-2 and MMP-7 was accomplished in living cells under single wavelength excitation. The apparent differences in the fluorescence imaging indicated distinct differences between the expression levels of MMPs between the human normal liver cells and the human hepatoma cells.     

Highly sensitive electrochemical label-free aptasensor based on dual electrocatalytic amplification of Pt-AuNPs and HRP

In this work, a label-free electrochemical aptamer-based sensor (aptasensor) was constructed on account of the direct immobilization of redox probes on an electrode surface. For this proposed aptasensor, a gold nanoparticles (AuNPs)-coated electrode was firstly modified with redox probes-nickel hexacyanoferrates nanoparticles (NiHCFNPs) through chemisorption and electrostatic adsorption. Then, platinum-gold alloy nanoparticles (Pt-AuNPs) and horseradish peroxidase (HRP) were respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the electrochemical signal of NiHCFNPs and immobilizing thiolated thrombin aptamers (TBAs). In the presence of target thrombin, the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of NiHCFNPs amplified by the Pt-AuNPs and HRP toward H(2)O(2). The proposed method avoided the redox probes labeling process, increased the amount of redox probes, and further amplified the electrochemical signal. Thus, the approach showed a high sensitivity and a wider linearity to thrombin in the range between 0.01 nM and 50 nM with a detection limit of 6.3 pM.     

A Sensitive Signal-off Electrochemical Aptasensor for Thrombin Detection using PB−Au@MoS2 Nanomaterial as Both Platform and Signal Reporter

Human serum is one of the effective samples for point-of-care testing (POCT). Sensitive and quick determination of thrombin content in human serum samples is important. An electrochemical aptasensor based on Prussian blue and Au nanoparticles loaded MoS₂ nanoflowers (PB−Au@MoS₂) hybrid was constructed. By using PB−Au@MoS₂ as both a substrate and a signal reporter, this aptasensor could demonstrate excellent performance for thrombin detection with a detection linear range from 0.01 pM to 30 nM and detection limit down to 1 fM. This work may provide a strategy to establish effective and sensitive sensing devices for thrombin in clinical diagnosis.     

Aptasensor for Thrombin Based on Carbon Nanotubes‐Methylene Blue Composites

The amperometric and EQCM aptasensors based on DNA aptamers immobilized by avidin‐biotin method or by electrostatic adsorption onto multiwalled carbon nanotube layer contained methylene blue (MB) have been developed and examined for thrombin detection in buffer and in spiked blood serum. The presence of MB increases the binding capacity of the surface layer and enhances the range of thrombin concentrations to be determined. This results in significant improvement of analytical characteristics of thrombin detection. The EQCM aptasensors allowed us to detect 0.3–100 nM and amperometric aptasensors 10–1000 nM of thrombin.     

基于无标记的核酸适配体荧光生物传感器检测凝血酶

设计了一个简单、通用、基于核酸适配体无标记的高敏感、高专一检测凝血酶的荧光方法。以无标记凝血酶核酸适配体单链DNA为识别元素,Pico Green染料传导互补双链的荧光信号。Pico Green是一种不对称菁,当其单独存在时不产生荧光信号,而当其被吸附到互补的双链DNA上时,可产生很强的荧光信号,但被吸附到单链DNA上时,却无明显的信号改变。基于该性质,将其用于凝血酶的检测。该方法对凝血酶的响应线性范围为1.0×10~(-14)~1.0×10~(-7)mol/L,相关系数(r~2)为0.99,检出限为1.0×10~(-14)mol/L。1.0×10~(-8)mol/L两种干扰物质(牛血清蛋白和细胞色素C)的存在不影响凝血酶的检测,表明该方法对凝血酶具有非常好的专一性。该方法成功应用于对人血清样品的检测,其平均回收率为97%~102%。方法可简单、灵敏、特异性地检测凝血酶,有望用于医学临床诊断等领域。     

Dual‐Luminophore‐Labeled Gold Nanoparticles with Completely Resolved Emission for the Simultaneous Imaging of MMP‐2 and MMP‐7 in Living Cells under Single Wavelength Excitation

Although considerable effort has been devoted to the design of various nanoprobes for the fluorescent detection of multiple biomarkers in a single assay, they often suffer from emission‐overlapping, owing to small Stokes shifts and wide emission spectra, which results in cross‐talk and inaccurate quantification. Herein, we report the design and synthesis of a new nanoprobe for multienzyme detection with completely resolved emission peaks under single‐wavelength excitation. The probe was assembled by attaching a cleavable peptide spacer, which was comprised from a matrix metalloproteinase‐2 (MMP‐2) substrate and a MMP‐7 substrate, onto the surface of gold nanoparticles (AuNPs) through cysteine residues. A lanthanide complex, BCTOT‐Eu^III (BCTOT=1,10‐bis(5′‐chlorosulfo‐thiophene‐2′‐yl)‐4,4,5,5,6,6,7,7‐octafluorodecane‐1,3,8,10‐tetraone), and 7‐amino‐4‐methylcoumarin (AMC) were attached to the N terminus and the C terminus of the peptide, respectively. In the presence of one or both targeting enzymes, the substrate was cleaved and fluorescence resonance energy transfer (FRET) between the dyes and AuNPs was prohibited, thereby resulting in the dramatic fluorescence emission of dyes. Importantly, there was no emission cross‐talk between the two dyes, thereby ensuring accurate detection of each enzyme. Based on this, the simultaneous fluorescence image of MMP‐2 and MMP‐7 was accomplished in living cells under single wavelength excitation. The apparent differences in the fluorescence imaging indicated distinct differences between the expression levels of MMPs between the human normal liver cells and the human hepatoma cells.     

An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin

The aptameric enzyme subunit (AES) is an artificial enzyme subunit that can allosterically control partner enzyme activity. By means of an AES, target molecules can be detected by measurement of enzymatic activity in a homogeneous solution. We have developed a thrombin aptamer-based AES that can detect several targets by measuring clotting time in a fibrinogen solution. Measurement of the clotting activity in the fibrinogen solution is not suitable for a convenient biosensor. However, kinetics measurement of clotting activity is suitable for convenient detection of thrombin activity. The interference-enhanced reflection (IER) method is a simple, real-time technique to detect the thickness and/or refractive index of a thin film formed on a glass substrate surface. We demonstrated that clotting activity on a glass substrate with immobilized thrombin can be monitored in real time by IER. By means of the IER method, we were able to detect the target molecules of an AES. IER-based sensors have already been commercialized as portable sensors for the detection of organic compounds. Thus, portable biosensors could be developed by combination of the IER and AES technologies.