Chemical-Chemical Redox Cycle Signal Amplification Strategy Combined with Dual Ratiometric Immunoassay for Surface-Enhanced Raman Spectroscopic Detection of Cardiac Troponin I.

Improving the sensitivity and reproducibility of surface-enhanced Raman spectroscopy (SERS) methods for the detection of bioactive molecules is crucial in biological process research and clinical diagnosis. Herein, we designed a novel SERS platform for cardiac troponin I (cTnI) detection by a chemical-chemical redox cycle signal amplification strategy combined with a dual ratiometric immunoassay. First, ascorbic acid (AA) was generated by enzyme-assisted immunoreaction with a cTnI-anchored sandwich structure. Then, oxidized 4-mercaptophenol (ox4-MP) was reacted with AA to produce 4-mercaptophenol (4-MP). Quantitative analysis of cTnI was realized by a Raman signal switch between ox4-MP and 4-MP. Specifically, AA could be regenerated by reductant (tris(2-carboxyethyl) phosphine, TCEP), which in turn produced more signal indicator 4-MP, causing significant signal amplification for cTnI analysis by SERS immunosensing. Moreover, a dual ratiometric-type SERS method was established with the intensity ratio I1077/I822 and I633/I822, which improved the reproducibility of the cTnI assay. The excellent performance of the chemical-chemical redox cycle strategy and ratio-type SERS assay endows the method with high sensitivity and reproducibility. The linear ranges of cTnI were 0.001 to 50.0 ng mL-1 with detection limits of 0.33 pg mL-1 (upon I1077/I822) and 0.31 pg mL-1 (upon I635/I822), respectively. The amount of cTnI in human serum samples yielded recoveries from 89.0 to 114%. This SERS method has remarkable analytical performance, providing an effective approach for the early diagnosis of cardiovascular diseases, and has great latent capacity in the sensitive detection of bioactive molecules.

A Highly Sensitive Catalytic Hairpin Assembly-Based Dynamic Light-Scattering Biosensors for Telomerase Detection in Bladder Cancer Diagnosis.

Precise evaluation of telomerase activity is highly crucial for early cancer diagnosis. In this study, a sensitive catalytic hairpin assembly-dynamic light scattering (CHA-DLS) assay for telomerase activity detection is developed by using the diameter change of gold nanoparticle (AuNP) probes. The telomerase substrate primer can be extended in the presence of telomerase, producing a telomerase extension product (TEP) with telomeric repeat units (TTAGGG)n at its 3'-end. The TEP can specifically trigger the CHA process and form tremendous AuNPs-H1/H2 nanostructures, resulting in a significant increase in the diameter measured by DLS. Telomerase activity from different cancer cell lines (MCF-7, Huh7, and 5637) was detected using the proposed strategy, the diameter of AuNP probes increased with the number of cancer cells, and this method can accurately detect telomerase activity down to 6 MCF-7 cells, 10 Huh7 cells, and three 5637 cells. Moreover, the CHA-DLS biosensor was successfully applied in urine specimens from healthy individuals and different cancer patients, which can distinguish bladder cancer patients from healthy people and other cancer patients, indicating that the noninvasive method has a great potential for application in early diagnosis of bladder cancer.

Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.

The incorporation of AuNPs into polymerase chain reaction (PCR) has become a promising strategy to develop sensitive sensing platforms, due to desirable optical properties of AuNPs and the exponential amplification power of PCR. However, the combination of AuNPs to PCR usually fails to reach expected sensitivity along with additional steps. Here we report a one-step and universal PCR-based biosensor for size-dependent detection of nucleic acids and proteins by using the dynamic light scattering (DLS) technique. This sensing system employs a DNA modified AuNPs (AuNPs-DNA) probe to serve as the TaqMan like signal probe, in which DNA on the surface of AuNPs can be cleaved by Taq polymerase during the extension process of PCR, causing the aggregation of AuNPs to be detected by DLS. This strategy enables sequence-specific recognition of target double-stranded DNA (dsDNA), overcoming the background signal change that triggered by the increase of the PCR cycle. It further demonstrates its applicability in detecting a foodborne pathogen Listeria monocytogenes with a detection limit of 1.2 fg µL-1, which is more sensitive than colorimetric methods. Moreover, by utilizing a proximity assay strategy, this biosensor shows the capability to the homogeneous detection of protein, showing a detection limit of 1.0 pM for a model thrombin. Together, the work demonstrates a reliable strategy to incorporate AuNPs into PCR amplification process as a signal probe, instead of the post-PCR process.

Ultrasensitive Detection of HIV DNA with Polymerase Chain Reaction-Dynamic Light Scattering.

Early diagnosis of HIV biomarkers or genes is the key to reducing acquired immunodeficiency syndrome (AIDS) mortality. In our work, we developed a novel polymerase chain reaction-dynamic light scattering (PCR-DLS) assay for one-step sensitive detection of HIV DNA based on the average-diameter change of gold nanoparticles (AuNPs). This is the first PCR assay that makes use of the DLS technique as a signal read-out, with the particle size measured by DLS increasing with the concentration of target DNA. With the help of the AuNP probes, this PCR-DLS assay can effectively improve the specificity of PCR reactions, which can greatly increase the detection sensitivity, with a detection limit of 1.8 aM (S/N = 3). In addition, the proposed strategy was successfully used to analyze target DNA in human serum samples, indicating that the PCR-DLS assay has a promising potential application for rapid and early clinical diagnosis of HIV infection.

A sensitive colorimetric DNA biosensor for specific detection of the HBV gene based on silver-coated glass slide and G-quadruplex-hemin DNAzyme.

A sensitive colorimetric DNA biosensor for specific detection of single stranded oligonucleotide (ssDNA) is proposed in this paper. The biosensor is based on silver-coated glass (SCGS) and G-quadruplex-hemin DNAzyme. Capture DNA is immobilized on the surface of SCGS by Ag-S bond. Signal DNA can be used to hybridize with the target DNA which is selected from the Hepatitis B virus(HBV) gene as target HBV DNA, and the HRP-mimicking G-quadruplex-hemin DNAzyme can be formed through the function of a guanine-rich fragment from signal DNA to catalyze the oxidation of 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonicacid (ABTS2- ) by H2 O2 . The reaction will be monitored along the side of absorbance changes at 418 nm and it can be viewed by naked eye with the change of color as well. Upon addition of target Hepatitis B virus(HBV) DNA, signal DNA could bind on the surface of SCGS, and the concentration of G-quadruplex-hemin DNAzyme immobilizing on the surface of SCGS is depended on that of target HBV DNA. Under the optimum conditions, the absorption was proportional to the concentration of target HBV DNA over the range from 0.5 to 100 nM, with a detection limit of 0.2 nM. In addition, the biosensor is target specific and practicability. This assay might open a new avenue for applying in the diagnosis of HBV disease in the future.

Determination of bacterial DNA based on catalytic oxidation of cysteine by G-quadruplex DNAzyme generated from asymmetric PCR: Application to the colorimetric detection of Staphylococcus aureus.

A one-step, one-tube colorimetric assay is described for the detection of bacterial double-stranded DNA (dsDNA). It utilizes a G-quadruplex DNAzyme produced by an asymmetric polymerase chain reaction (As-PCR) that catalyzes the oxidation of cysteine to form cystine. This results in the formation of oligonucleotide-modified gold nanoparticles via triplex formation, and eventually in a color change from red to blue that occurs within about 10 mins. This can be measured by ratiometric colorimetric (at 525 and 600 nm). The limit of detection (LOD) for the model analyte (dsDNA of Staphylococcus aureus (S. aureus)) is as low as 0.28 pg per 0.05 mL with a good linear response ranging from 16.0 fg·µL-1 to 1.6 ng·µL-1. This is much lower than previously reported LODs. The assay is highly selective for S. aureus dsDNA over a range of other bacterial DNAs. Conceivably, it provides an attractive alternative tool for rapid detection of bacterial dsDNA as required in pathogen screening in the food industry. Graphical abstract Schematic presentation of a colorimetric assay for bacterial DNA. It is based on the catalytic activity of a G-quadruplex DNAzyme that is formed by an asymmetric PCR involving triplex DNA formation and gold nanoparticle (AuNPs) aggregation.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

Fluorescence recognition of double-stranded DNA based on the quenching of gold nanoparticles to a fluorophore labeled DNA probe.

An ultrasensitive fluorescent platform for sequence-specific recognition of double-stranded DNA (dsDNA) based on the quenching of gold nanoparticles (AuNPs) to a fluorophore labeled DNA probe was developed. The target dsDNA could hybridize with the loop portion of the molecular beacon (MB) to form a triplex DNA structure and opened the "stem-loop" structure of the MB; such triplex DNA was used as an assistant probe (AP). Meanwhile, a fluorophore labeled DNA-AuNP probe that contained a specific enzyme cleavage site was introduced and its fluorescence signal was efficiently quenched due to the vicinity of the fluorophore to the AuNP surface. Such a DNA-AuNP probe could hybridize with the 5' stem portion of the MB in the AP to form duplex DNA strands that contained a specific enzyme cleavage site for the nicking enzyme assisted cleavage reaction, and resulted in the release of the fluorophore from the AuNP surface and the recovery of the fluorescence signal. Because the AP remains intact during such a cleavage process, it could be reused to hybridize with the next DNA-AuNP probe and trigger the nicking nuclease assisted signal amplification. Under optimal conditions, a low detection limit of 3.8 pM was obtained for dsDNA detection, and the assay has high sequence specificity for dsDNA detection.

Detection of Glucose with Atomic Absorption Spectroscopy by Using Oligonucleotide Functionalized Gold Nanoparticle.

A novel method for the detection of glucose was established with atomic absorption spectroscopy by using the label of gold nanoparticle (AuNP). Silver-coated glass assembled with oligonucleotide 5'-SH-T12-AGA CAA GAG AGG-3' (Oligo 1) was acted as separation probe, oligonucleotide 5'-CAA CAG AGA ACG-T12-SH-3' modified gold nanoparticle (AuNP-Oligo 2) was acted as signal-reporting probe. Oligonucleotide 5'-CGT TCT CTG TTG CCT CTC TTG TCT-3' (Oligo 3) could hybridize with Oligo 1 on the surface of silver-coated glass and AuNP-Oligo 2, and free AuNP-Oligo 2 could be removed by rinsing with buffer. Hence the concentration of Oligo 3 was transformed into the concentration of gold element. In addition, Oligo 3 could be cleaved into DNA fragments by glucose, glucose oxidase and Fe(2+)-EDTA through Fenton reaction. Thereby the concentration of glucose could be transformed to the absorbance of gold element. Under the optimum conditions, the integrated absorbance decreased proportionally to the concentration of glucose over the range from 50.0 µM to 1.0 mM with a detection limit of 40.0 µM. Moreover, satisfactory result was obtained when the assay was used to determinate glucose in human serum.

Triplex DNA logic gate based upon switching on/off their structure by Ag(+)/cysteine.

The formation of intramolecular triplex DNA can be regulated by Ag(+) and Cys (cysteine), which switch off/on the fluorescence of the oligonucleotides, 5'-TAMRA-TTC TCT TCC TCT TCC TTC TGA CGA CAG TTG ACT CTT CCT TCT CCT TCT CTT-BHQ-2-3' (Oligo 1) and 3'-GAA GGA AGA GGA AGA GAA-5' (Oligo 2). Based on this principle, sensors for Ag(+) and Cys are developed. The sensor for Ag(+) has a linear range of 2.5 nM-40 nM and a detection limit of 1.8 nM, whereas the sensor for Cys has a linear range of 10.0 nM-120.0 nM and a detection limit of 8.2 nM. Furthermore, the fluorescence is reversible with the alternate addition of Ag(+) and Cys. We constructed a DNA logic gate using Ag(+) and Cys as the input, and the fluorescence intensity as the output. The DNA logic gate is simple; moreover, it has a fast response and good reversibility.

Localized surface plasmon resonance light-scattering sensor for mercury(II) ion with label-free gold nanoparticles.

A localized surface plasmon resonance (LSPR) light-scattering sensor for Hg2+ was developed with unmodified gold nanoparticles (AuNPs) based upon the specific recognition property of Hg2+ with T-T mismatched base pair. Oligonucleotide 5'-GTTCTTTGTCTTCA-3'(oligo-1) and 5'-TGTAGTCTATGTAC-3'(oligo-2) can adsorb on the surface of AuNPs, which can prevent them from aggregation because of electrostatic repulsion. However, the DNA hybridization occurred between oligo-1 and oligo-2 upon addition of Hg2+, can induce the desorption of oligonucleotide from the surface of AuNPs, and trigger the aggregation of AuNPs accompany with the increase of LSPR light-scattering intensity. Under the optimum conditions, the intensity was proportional to the concentration of Hg2+ over the range 53.1-530 nM, and the detection limit was 29.4 nM.

Detection of Pb²âº at attomole levels by using dynamic light scattering and unmodified gold nanoparticles.

Lead ion (Pb²âº) accumulation in nature can affect the environment and human health severely. Thus, rapid and sensitive detection is of great importance. One-step detection of Pb²âº at attomole levels was realized by using dynamic light scattering (DLS) technique coupled with unmodified gold nanoparticles (AuNPs). Pb²âº-dependent DNAzyme was double-stranded and could not adsorb on the surface of AuNPs, while the substrate strand could be cleaved into ssDNA fragments on addition of Pb²âº. The ssDNA fragments could adsorb on the surface of AuNPs and prevent them from aggregating in the presence of NaCl. Therefore, the disperse state of AuNPs changed on addition of Pb²âº in the presence of DNAzyme and NaCl, which was estimated with an average hydrodynamic diameter by using DLS. Under optimum conditions, the average diameter of the solution decreased linearly with the concentration of Pb²âº over the range from 10 to 300 pM, with a detection limit of 6.2 pM. Moreover, satisfactory results were obtained when the proposed method was applied in the detection of Pb²âº in water samples.

A highly sensitive sensor for Cu2+ with unmodified gold nanoparticles and DNAzyme by using the dynamic light scattering technique.

Copper ion (Cu(2+)) plays an important role in many biological reactions, and a suitable level of Cu(2+) is necessary for the regular metabolism of life. Thus developing a sensitive and simple method for determination of Cu(2+) is essential. Here, a novel and sensitive Cu(2+) sensor was developed based on detecting the average hydrodynamic diameter of AuNPs by using dynamic light scattering (DLS). Cu(2+)-specific DNAzyme was double-strand and could not adsorb on the surface of AuNPs, accordingly AuNPs aggregation would occur with the addition of NaCl. However, Cu(2+) could cleave DNAzyme and release single-stranded DNA (ssDNA) fragments, which could adsorb on the surface of AuNPs and prevent them from aggregation. Such differences in DNA adsorption ability on AuNPs before and after the addition of Cu(2+) affected the disperse state of AuNPs directly, and then affected their average hydrodynamic diameter, which could be detected with the DLS technique. Based upon the above mentioned principle, detection of Cu(2+) could be realized over the range from 100 pM to 2.0 nM, with a linear regression equation of D = 306.73 - 89.66C (C: nM, R = 0.9953) and a detection limit of 60 pM (3δ/slope). Moreover, satisfactory results were obtained when the assay was applied in the detection of Cu(2+) in water samples.

Sensitive detection of H2O2 and H2O2-related reactant with Ru(bipy)(2)(7,8-dimethyl-dipyridophenazine)2+ and oligodeoxyribonucleotide.

A sensor for H(2)O(2) and H(2)O(2)-related reactant was constructed with oligonucleotides and Ru(bipy)(2)dppx(2+) (bipy = 2,2'-bipyridine, dppx = 7,8-dimethyl-dipyridophenazine), which was performed by converting the H(2)O(2)-induced DNA cleavage into the change of luminescence. The 'DNA light switch' Ru(bipy)(2)dppx(2+) could emit strong luminescence in the presence of dsDNA. DNA cleavage occurred upon addition of H(2)O(2) due to the Fenton reaction, which resulted in the decrease of the luminescence of Ru(bipy)(2)dppx(2+). Therefore, the luminescence intensity depended on the concentration of H(2)O(2) and H(2)O(2)-related reactants, and the detection limits for H(2)O(2), uric acid and cholesterol were 0.20 µM, 0.46 µM and 1.25 µM, respectively. The recovery varied between 94.0% and 105.0% when the assay was applied to the determination of uric acid and cholesterol in biological samples, which demonstrated the good practicability of the assay.

RGB color analysis of formaldehyde in vegetables based on DNA functionalized gold nanoparticles and triplex DNA.

A highly sensitive and selective RGB color analysis for the detection of formaldehyde (FA) was developed by using a DNA functionalized gold nanoparticle (AuNPs-DNA) probe. When complementary oligonucleotides (oligo 2 and oligo 3) and a silver ion (Ag+) were added to the AuNPs-DNA solution, triplex DNA was formed, resulting in the aggregation of AuNPs, and accompanied by a solution color change from red to purple. With the addition of formaldehyde, it reacted with Ag+, decreased the stability of triplex DNA between AuNPs-DNA, induced the dispersion of AuNPs, and the color of AuNPs recovered to red. Therefore, the formaldehyde concentration could be estimated with the RGB (red, green, blue) values of the AuNP solution by using a smartphone application (APP). The R value of the system was proportional to the concentration of formaldehyde within the range of 0.23-4.50 mg L-1, with a detection limit of 0.14 mg L-1. The method has been successfully applied to detect the residues of formaldehyde in vegetable samples and has the potential of the on-site determination of formaldehyde.

Hybridization chain reaction and DNAzyme-based dual signal amplification strategy for sensitive fluorescent sensing of aflatoxin B1 by using the pivot of triplex DNA.

This work developed an enzyme-free fluorescent aptasensor for sensitive aflatoxin B1 (AFB1) detection based on a dual signal amplification strategy of hybridization chain reaction (HCR) and Zn2+-dependent DNAzyme. In the presence of AFB1, the aptamer specifically binds to the target, releasing the blocking DNA, which can initiate HCR between hairpin probes H1 and H2. With the addition of the substrate strand (Zn-Sub) and enzyme strand (Zn-Enz) of DNAzyme, HCR product can hybridize with Zn-Sub and Zn-Enz to form triplex DNA and Y-shaped structure together, which further activates the DNAzyme to cleave Zn-Sub. Then, two separated fragments of Zn-Sub respectively hybridize with the fluorescent probe and quencher probe, which results in a dramatic increase in the fluorescence intensity. The proposed aptasensor shows high sensitivity and selectivity for AFB1 detection with a detection limit of 0.22 nmol/L in a linear range of 0.4-16 nmol/L. Moreover, the fluorescent aptasensor exhibits acceptable applicability for detecting AFB1 in oil samples with satisfactory recoveries of 92.2-107.8%. Results are also in agreement with those of the ELISA method, indicating that the fluorescent sensing strategy has great potential applications in food safety control.

Nicking enzyme-free strand displacement amplification-assisted CRISPR-Cas-based colorimetric detection of prostate-specific antigen in serum samples.

Immunosorbent assay is the gold standard diagnostic technique for the detection of protein biomarkers. However, this technique tends to have low sensitivity and requires laborious manipulation. Although advanced CRISPR-Cas-based biosensors offer advantages of simplicity, low cost and high accuracy, the synergy of using CRISPR-Cas-assisted dual signal amplification system for rapid diagnosis of protein biomarkers remains scarce. In this work, we report a synergetic signal amplification system comprising CRISPR-Cas12a and nicking enzyme-free strand displacement amplification (SDA) technique for accurate detection of prostate-specific antigen (PSA). The presence of PSA will initiate the nicking enzyme-free SDA process, generating amplicons that can be recognized by the CRISPR-Cas12a system. The activated CRISPR-Cas system will then mediate trans-ssDNA cleavage of neighboring linker DNA, which unlocks the gold nanoparticles (AuNPs) signal probes and gives a distance-dependent colorimetric readout. This assay could detect PSA in aqueous buffer sensitively and selectively with a limit of detection (LOD) down to 0.030 ng mL-1. Importantly, this assay was successfully applied for discriminating four blood samples from prostate cancer patients among thirteen blood samples from normal individuals/cancer patients accurately. This work will open an avenue for the development of SDA-CRISPR-AuNPs hybrid sensing systems, offering great potential for the development of non-invasive point-of-care diagnostic tools for prostate cancer.

Dynamic-light-scattering-based sequence-specific recognition of double-stranded DNA with oligonucleotide-functionalized gold nanoparticles.

An ultrasensitive and simple dynamic-light-scattering (DLS) assay for the sequence-specific recognition of double-stranded DNA (dsDNA) was developed based on detection of the average diameter change of Au nanoparticle (AuNP) probes modified with oligonucleotides 5'-TTTCTCTTCCTT- CTCTTC-(T)(12)-SH-3' (Oligo 1) and 5'-TTCTTTCTTTTCTTTTTC-(T)(12)- SH-3' (Oligo 2). The target dsDNA was composed of two complementary oligonucleotides: 5'-AAAGAGAAGGAAGAGAAGAAGAAAGAAAAGAAAAAG-3' (Oligo 3) and 3'-TTTCTCTTCCTTCTCTTCTTCTTTCTTTTCTTTTTC-5' (Oligo 4). Hybridization of the two AuNPs-Oligo probes with the target dsDNA induced aggregation of the target dsDNA by forming triplex DNA, which accordingly increased the average diameter. This diameter change could then be detected by DLS. The average diameter was proportional to the target dsDNA concentration over the range from 593 fM to 40 pM, with a detection limit of 593 fM. Moreover, the assay had good sequence specificity for the target dsDNA.

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide.

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.

[Spectral study on the interaction of [Cu(DPPZ)(L-Ser)]+ complex with DNA and its analytical application].

The characteristics of resonance light scattering (RLS), UV-visible absorption spectra and fluorescence spectra of [Cu(DPPZ)(L-Ser)]+ with DNA were studied and a RLS method for the determination of DNA was established. [Cu(DPPZ) (L-Ser)]+ could assemble on the surface of DNA through intercalation, and enhanced the RLS of DNA in the tris buffer of pH 7. 2. The maximum resonance light scattering peak appeared at 400 nm. Under the optimum conditions, the enhanced intensity of RLS was proportional to the concentration of DNA over the range of 0.42 - 4.2 ng x mL(-1), with a detection limit (3sigma/k) of 0.29 ng x mL(-1). The method was used for the determination of DNA samples with the recoveries between 97.8% and 106%.