A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation

Background DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy. Methods A novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex. Results The obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10−15 to 10−7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples. Conclusion This strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

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DNAzyme-Functionalized R-Phycoerythrin as a Cost-Effective and Environment-Friendly Fluorescent Biosensor for Aqueous Pb2+ Detection

Sensors ◽

10.3390/s19122732 ◽

2019 ◽

Vol 19 (12) ◽

pp. 2732 ◽

Cited By ~ 3

Author(s):

Jikui Wu ◽

Yunfei Lu ◽

Ningna Ren ◽

Min Jia ◽

Ruinan Wang ◽

...

Keyword(s):

Health Care ◽

Food Safety ◽

Lake Water ◽

Limit Of Detection ◽

Cost Effective ◽

Fluorescence Signal ◽

Environment Friendly ◽

Substrate Complex ◽

Fluorescent Biosensor ◽

Excellent Selectivity

The sensitive detection of Pb2+ is of significant importance for food safety, environmental monitoring, and human health care. To this end, a novel fluorescent biosensor, DNAzyme-functionalized R-phycoerythrin (DNAzyme-R-PE), was presented for Pb2+ analysis. The biosensor was prepared via the immobilization of Iowa Black® FQ-modified DNAzyme–substrate complex onto the surface of SPDP-functionalized R-PE. The biosensor produced a minimal fluorescence signal in the absence of Pb2+. However, Pb2+ recognition can induce the cleavage of substrate, resulting in a fluorescence restoration of R-PE. The fluorescence changes were used to measure sensitively Pb2+ and the limit of detection was 0.16 nM with a linear range from 0.5–75 nM. Furthermore, the proposed biosensor showed excellent selectivity towards Pb2+ even in the presence of other metal ions interferences and was demonstrated to successfully determine Pb2+ in spiked lake water samples.

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A Ratiometric Fluorescent Aptamer Homogeneous Biosensor Based on Hairpin Structure Aptamer for AFB1 Detection

10.21203/rs.3.rs-1136909/v1 ◽

2022 ◽

Author(s):

Beibei Feng ◽

Fei Zhao ◽

Min Wei ◽

Yong Liu ◽

Xinyu Ren ◽

...

Keyword(s):

Limit Of Detection ◽

Resonance Energy Transfer ◽

Fluorescence Emission ◽

Resonance Energy ◽

Hairpin Structure ◽

Fluorescence Signal ◽

Detection Range ◽

Quenching Effect ◽

Double Stranded Dna ◽

Aflatoxin B

Abstract On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescent aptamer sensor demonstrates a weak fluorescence emission of Cy3 and a high fluorescence emission of Cy5 due to the quenching effect of BHQ2. The double-stranded DNA structure will be disintegrated in the presence of AFB1, resulting the removal of Cy3 and the close of Cy5 with BHQ2. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, indicating it had good application potential in practical samples.

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Design and Quantitative Analysis of Cancer Detection System Based on Fluorescence Immune Analysis

Journal of Healthcare Engineering ◽

10.1155/2019/1672940 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Lei Shao ◽

Longyu Zhang ◽

Shilin Li ◽

Pengyuan Zhang

Keyword(s):

Human Blood ◽

High Efficiency ◽

Limit Of Detection ◽

Detection System ◽

Quantitative Result ◽

Threshold Function ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Detection Accuracy ◽

Detection Range

Human blood is an important medical detection index. With the development in clinical medical detection instruments and detection technology, the requirements for detection accuracy and efficiency have been gradually improved. Fluorescent immunochromatography is a new detection technique. It has the characteristics of high efficiency, convenience, no pollution, and wide detection range. Human blood can be detected quickly using fluorescent immunochromatography. At present, it has received great attention from the field of clinical testing. In this paper, a set of fluorescent immunochromatographic analyzer has been designed. It is mainly based on the principle of fluorescence immunochromatography. A new method of signal analysis and system design for fluorescent immunochromatography analyzer is proposed. By using the improved threshold function denoising algorithm, the quantitative detection of fluorescent immunochromatographic strip is realized. The concentration of pathogenic factors (cancer cells) in human serum can be measured conveniently and accurately. The system integrates many peripheral modules, including fluorescence signal acquisition, fluorescence signal processing, quantitative curve fitting, and test results. In this paper, the quantitative detection experiments of the system are carried out in three aspects: linearity, repeatability, and sensitivity. The experimental results show that the linear correlation coefficient is up to 0.9976, and the limit of detection is up to 0.05 ng/ml. The requirements of the system are satisfied. The system performance is good, and the quantitative result is accurate. Therefore, the establishment of a fluorescence analysis system is of great significance.

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A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis

Nature Communications ◽

10.1038/s41467-021-23185-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Guanhua Xun ◽

Stephan Thomas Lane ◽

Vassily Andrew Petrov ◽

Brandon Elliott Pepa ◽

Huimin Zhao

Keyword(s):

Limit Of Detection ◽

Low Cost ◽

High Volume ◽

N Gene ◽

Testing System ◽

Target Sequence ◽

One Pot ◽

Sequence Detection ◽

Highly Sensitive ◽

Speed Accuracy

AbstractThe need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

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A Compact Detection Platform Based on Gradient Guided-Mode Resonance for Colorimetric and Fluorescence Liquid Assay Detection

Sensors ◽

10.3390/s21082797 ◽

2021 ◽

Vol 21 (8) ◽

pp. 2797

Author(s):

Jing-Jhong Gao ◽

Ching-Wei Chiu ◽

Kuo-Hsing Wen ◽

Cheng-Sheng Huang

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Fluorescence Emission ◽

Assay Sensitivity ◽

Detection Range ◽

Current Form ◽

Guided Mode ◽

Spectral Detection ◽

Guided Mode Resonance ◽

Colorimetric Assays

This paper presents a compact spectral detection system for common fluorescent and colorimetric assays. This system includes a gradient grating period guided-mode resonance (GGP-GMR) filter and charge-coupled device. In its current form, the GGP-GMR filter, which has a size of less than 2.5 mm, can achieve a spectral detection range of 500–700 nm. Through the direct measurement of the fluorescence emission, the proposed system was demonstrated to detect both the peak wavelength and its corresponding intensity. One fluorescent assay (albumin) and two colorimetric assays (albumin and creatinine) were performed to demonstrate the practical application of the proposed system for quantifying common liquid assays. The results of our system exhibited suitable agreement with those of a commercial spectrometer in terms of the assay sensitivity and limit of detection (LOD). With the proposed system, the fluorescent albumin, colorimetric albumin, and colorimetric creatinine assays achieved LODs of 40.99 and 398 and 25.49 mg/L, respectively. For a wide selection of biomolecules in point-of-care applications, the spectral detection range achieved by the GGP-GMR filter can be further extended and the simple and compact optical path configuration can be integrated with a lab-on-a-chip system.

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Ratiometric Fluorescent Biosensors for Glucose and Lactate Using an Oxygen-Sensing Membrane

Biosensors ◽

10.3390/bios11070208 ◽

2021 ◽

Vol 11 (7) ◽

pp. 208

Author(s):

Hong Dinh Duong ◽

Jong Il Rhee

Keyword(s):

Limit Of Detection ◽

Oxygen Sensing ◽

High Sensitivity ◽

Glucose Sensing ◽

Oxidation Reactions ◽

Lactate Oxidase ◽

Lactate Oxidation ◽

Detection Range ◽

Quenching Effect ◽

Sensing Membrane

In this study, ratiometric fluorescent glucose and lactate biosensors were developed using a ratiometric fluorescent oxygen-sensing membrane immobilized with glucose oxidase (GOD) or lactate oxidase (LOX). Herein, the ratiometric fluorescent oxygen-sensing membrane was fabricated with the ratio of two emission wavelengths of platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) doped in polystyrene particles and coumarin 6 (C6) captured into silica particles. The operation mechanism of the sensing membranes was based on (i) the fluorescence quenching effect of the PtP dye by oxygen molecules, and (ii) the consumption of oxygen levels in the glucose or lactate oxidation reactions under the catalysis of GOD or LOX. The ratiometric fluorescent glucose-sensing membrane showed high sensitivity to glucose in the range of 0.1–2 mM, with a limit of detection (LOD) of 0.031 mM, whereas the ratiometric fluorescent lactate-sensing membrane showed the linear detection range of 0.1–0.8 mM, with an LOD of 0.06 mM. These sensing membranes also showed good selectivity, fast reversibility, and stability over long-term use. They were applied to detect glucose and lactate in artificial human serum, and they provided reliable measurement results.

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A highly sensitive octopus-like azobenzene fluorescent probe for determination of abamectin B1 in apples

Scientific Reports ◽

10.1038/s41598-021-84221-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhenlong Guo ◽

YiFei Su ◽

Kexin Li ◽

MengYi Tang ◽

Qiang Li ◽

...

Keyword(s):

Fluorescent Probe ◽

Limit Of Detection ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Ionization Mass ◽

Limit Of Quantification ◽

Visible Absorption ◽

Esi Ms ◽

Relative Standard ◽

Ft Ir

AbstractThe development of detecting residual level of abamectin B1 in apples is of great importance to public health. Herein, we synthesized a octopus-like azobenzene fluorescent probe 1,3,5-tris (5′-[(E)-(p-phenoxyazo) diazenyl)] benzene-1,3-dicarboxylic acid) benzene (TPB) for preliminary detection of abamectin B1 in apples. The TPB molecule has been characterized by ultraviolet–visible absorption spectrometry, 1H-nuclear magnetic resonance, fourier-transform infrared (FT-IR), electrospray ionization mass spectroscopy (ESI-MS) and fluorescent spectra. A proper determination condition was optimized, with limit of detection and limit of quantification of 1.3 µg L−1 and 4.4 μg L−1, respectively. The mechanism of this probe to identify abamectin B1 was illustrated in terms of undergoing aromatic nucleophilic substitution, by comparing fluorescence changes, FT-IR and ESI-MS. Furthermore, a facile quantitative detection of the residual abamectin B1 in apples was achieved. Good reproducibility was present based on relative standard deviation of 2.2%. Six carboxyl recognition sites, three azo groups and unique fluorescence signal towards abamectin B1 of this fluorescent probe demonstrated reasonable sensitivity, specificity and selectivity. The results indicate that the octopus-like azobenzene fluorescent probe can be expected to be reliable for evaluating abamectin B1 in agricultural foods.

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Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection

Chemosensors ◽

10.3390/chemosensors9030049 ◽

2021 ◽

Vol 9 (3) ◽

pp. 49

Author(s):

Pushap Raj ◽

Man Hwan Oh ◽

Kyudong Han ◽

Tae Yoon Lee

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Limit Of Detection ◽

Bacterial Pathogens ◽

Cost Effective ◽

Covalent Immobilization ◽

Label Free ◽

Self Assembled Monolayer ◽

Detection Range ◽

Linear Detection

Bacterial infections have become a significant challenge in terms of public health, the food industry, and the environment. Therefore, it is necessary to address these challenges by developing a rapid, cost-effective, and easy-to-use biosensor for early diagnosis of bacterial pathogens. Herein, we developed a simple, label-free, and highly sensitive immunosensor based on electrochemical detection using the Au@MoS₂–PANI nanocomposite. The conductivity of the glassy carbon electrode is greatly enhanced using the Au@MoS₂–PANI nanocomposite and a self-assembled monolayer of mercaptopropionic acid on the gold nanoparticle surface was employed for the covalent immobilization of antibodies to minimize the nonspecific adsorption of bacterial pathogens on the electrode surface. The biosensor established a high selectivity and sensitivity with a low limit of detection of 10 CFU/mL, and detected Escherichia coli within 30 min. Moreover, the developed biosensor demonstrated a good linear detection range, practical utility in urine samples, and electrode regenerative studies.

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Fluorescence Based on Surface Plasmon Coupled Emission for Ultrahigh Sensitivity Immunoassay of Cardiac Troponin I

Biomedicines ◽

10.3390/biomedicines9050448 ◽

2021 ◽

Vol 9 (5) ◽

pp. 448

Author(s):

Vien Thi Tran ◽

Heongkyu Ju

Keyword(s):

Surface Plasmon ◽

Cardiac Troponin ◽

Troponin I ◽

Limit Of Detection ◽

Delayed Diagnosis ◽

Cardiac Troponin I ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Label Free ◽

Ultrahigh Sensitivity

This work demonstrates the quantitative assay of cardiac Troponin I (cTnI), one of the key biomarkers for acute cardiovascular diseases (the leading cause of death worldwide) using the fluorescence-based sandwich immune reaction. Surface plasmon coupled emission (SPCE) produced by non-radiative coupling of dye molecules with surface plasmons being excitable via the reverse Kretschmann format is exploited for fluorescence-based sandwich immunoassay for quantitative detection of cTnI. The SPCE fluorescence chip utilizes the gold (2 nm)-silver (50 nm) bimetallic thin film, with which molecules of the dye Alexa 488 (conjugated with detection antibodies) make a near field coupling with the plasmonic film for SPCE. The experimental results find that the SPCE greatly improves the sensitivity via enhancing the fluorescence signal (up to 50-fold) while suppressing the photo-bleaching, permitting markedly enhanced signal-to-noise ratio. The limit of detection of 21.2 ag mL−1 (atto-gram mL−1) is obtained, the lowest ever reported to date amid those achieved by optical technologies such as luminescence and label-free optical sensing techniques. The features discovered such as ultrahigh sensitivity may prompt the presented technologies to be applied for early diagnosis of cTnI in blood, particularly for emergency medical centers overloaded with patients with acute myocardial infarction who would suffer from time-delayed diagnosis due to insufficient assay device sensitivity.

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Gold-nanourchin complexed silicon dioxide-probe on gap-fingered interdigitated electrode surface for Parkinson’s Disease determination by current–volt measurement

Nanomaterials and Nanotechnology ◽

10.1177/1847980420987352 ◽

2021 ◽

Vol 11 ◽

pp. 184798042098735

Author(s):

Xiaohong Li ◽

Wei Shi ◽

Wenyan Zhang ◽

Weiyao Chen ◽

Dan Cao ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Silicon Dioxide ◽

Limit Of Detection ◽

Detection System ◽

Interdigitated Electrode ◽

Limit Of Quantification ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Detection Range

Parkinson’s disease (PD) is a nervous disorder, affects physical movement, and leads to difficulty in balancing, walking, and coordination. A novel sensor is mandatory to determine PD and monitor the progress of the treatment. Neurofilament light chain (NfL) has been recognized as a good biomarker for PD and also helps to distinguish between PD and atypical PD syndromes. Immunosensor was generated by current–volt measurement on gap-fingered interdigitated electrode with silicon dioxide surface to determine NfL level. To enhance the detection, anti-NfL antibody was complexed with gold-nanourchin and immobilized on the sensing electrode. The current–volt response was gradually increased at the linear detection range from 100 fM to 1 nM. Limit of detection and sensitivity were 100 fM with the signal-to-noise ratio at n = 3 on a linear curve ( y = 0.081 x + 1.593; R 2 = 0.9983). Limit of quantification falls at 1 pM and high performance of the sensor was demonstrated by discriminating against other neurogenerative disease markers, in addition, it was reproducible even in serum-spiked samples. This method of detection system aids to measure the level of NfL and leads to determine the condition with PD.

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