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Guo J, Liu R, Ge Y, Fang R, Wang Y, Liu Y, Jiang H, Tang L, Feng J, Cao F, Zeng H. QCM biosensor based on silver aggregates and DNA ligase for ultrasensitive detection of BRAF-V600E single mutant gene in melanoma. Talanta 2025; 292:127997. [PMID: 40154042 DOI: 10.1016/j.talanta.2025.127997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2024] [Revised: 03/18/2025] [Accepted: 03/20/2025] [Indexed: 04/01/2025]
Abstract
The detection of BRAF-V600E gene mutations plays a pivotal role in the diagnosis, prediction, and therapeutic intervention of melanoma. In this work, a quartz crystal microbalance (QCM) biosensor was developed to detect the BRAF-V600E point mutation. The gold electrode of the QCM was incubated with a thiol (-SH) modified capture probe, subsequently treated with polyethylene glycol (PEG) to obstruct nonspecific binding sites on the electrode. Subsequently, the target sequence BRAF-V600E (alternatively referred to as BRAF) along with the detection probe were incubated to construct a functionalized layer. T4 DNA ligase was introduced to facilitate the connection between the detection and capture probes, thereby forming either elongated or abbreviated DNA chains. Ultimately, silver aggregates (AgAs) were formed and bound to DNA bases and exert a significant influence on the sensor's frequency. In instances where the target sequence is BRAF-V600E, the frequency shift is markedly higher in comparison to BRAF, thereby facilitating a clear distinction between the two. The developed biosensor exhibited a linear detection range of 1 pM-10 nM for BRAF-V600E, with a detection limit of 0.73 pM and an R2 value of 0.9923. The accuracy of the QCM method was rigorously validated through a comparative analysis with quantitative polymerase chain reaction (qPCR). This study holds significant implications for the early detection and precise of melanoma.
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Affiliation(s)
- Jia Guo
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Ruming Liu
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Yanghao Ge
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Ruiqin Fang
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China.
| | - Yixuan Wang
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Yan Liu
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Hanjia Jiang
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Lixia Tang
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Juan Feng
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China
| | - Fei Cao
- Oncology Department, The First Affiliated Hospital of Chengdu Medical College, Chengdu, 610500, PR China; Clinical Medical School, Chengdu Medical College, Chengdu, 610500, PR China; Key Clinical Specialty of Sichuan Province (Oncology Department), Chengdu, 610500, PR China
| | - Hongjuan Zeng
- School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, PR China.
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Lu Y, Xia W, Miao S, Wang M, Wu L, Xu T, Wang F, Xu J, Mu Y, Zhang B, Pan S. Clinical Characteristics of Severe COVID-19 Patients During Omicron Epidemic and a Nomogram Model Integrating Cell-Free DNA for Predicting Mortality: A Retrospective Analysis. Infect Drug Resist 2023; 16:6735-6745. [PMID: 37873032 PMCID: PMC10590600 DOI: 10.2147/idr.s430101] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Accepted: 10/13/2023] [Indexed: 10/25/2023] Open
Abstract
Objective This study aimed to investigate the clinical characteristics and risk factors of death in severe coronavirus disease 2019 (COVID-19) during the epidemic of Omicron variants, assess the clinical value of plasma cell-free DNA (cfDNA), and construct a prediction nomogram for patient mortality. Methods The study included 282 patients with severe COVID-19 from December 2022 to January 2023. Patients were divided into survival and death groups based on 60-day prognosis. We compared the clinical characteristics, traditional laboratory indicators, and cfDNA concentrations at admission of the two groups. Univariate and multivariate logistic analyses were performed to identify independent risk factors for death in patients with severe COVID-19. A prediction nomogram for patient mortality was constructed using R software, and an internal validation was performed. Results The median age of the patients included was 80.0 (71.0, 86.0) years, and 67.7% (191/282) were male. The mortality rate was 55.7% (157/282). Age, tracheal intubation, shock, cfDNA, and urea nitrogen (BUN) were the independent risk factors for death in patients with severe COVID-19, and the area under the curve (AUC) for cfDNA in predicting patient mortality was 0.805 (95% confidence interval [CI]: 0.713-0.898, sensitivity 81.4%, specificity 75.6%, and cut-off value 97.67 ng/mL). These factors were used to construct a prediction nomogram for patient mortality (AUC = 0.856, 95% CI: 0.814-0.899, sensitivity 78.3%, and specificity 78.4%), C-index was 0.856 (95% CI: 0.832-0.918), mean absolute error of the calibration curve was 0.007 between actual and predicted probabilities, and Hosmer-Lemeshow test showed no statistical difference (χ2=6.085, P=0.638). Conclusion There was a high mortality rate among patients with severe COVID-19. cfDNA levels ≥97.67 ng/mg can significantly increase mortality. When predicting mortality in patients with severe COVID-19, a nomogram based on age, tracheal intubation, shock, cfDNA, and BUN showed high accuracy and consistency.
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Affiliation(s)
- Yanfei Lu
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Wenying Xia
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Shuxian Miao
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Min Wang
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Lei Wu
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Ting Xu
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Fang Wang
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Jian Xu
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Yuan Mu
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Bingfeng Zhang
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
| | - Shiyang Pan
- Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China
- National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
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Liu JP, Zhang SC, Pan SY. Value of dynamic plasma cell-free DNA monitoring in septic shock syndrome: A case report. World J Clin Cases 2020; 8:200-207. [PMID: 31970188 PMCID: PMC6962064 DOI: 10.12998/wjcc.v8.i1.200] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/02/2019] [Revised: 11/18/2019] [Accepted: 11/30/2019] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Mortality due to septic shock is relatively high. The dynamic monitoring of plasma cell-free DNA (cfDNA) can guide the treatment of septic shock.
CASE SUMMARY Herein, we present a typical case of septic shock syndrome caused by the bacilli Acinetobacter baumannii and Pantoea. The patient complained of abdominal pain, fever and chills upon admission to the Emergency Department. Marked decreases in white blood cells and procalcitonin (PCT) were observed after the patient received continuous renal replacement and extracorporeal membrane oxygenation. Plasma cfDNA levels were consistently high, peaking at 1366.40 ng/mL, as measured by a duplex real-time PCR assay with an internal control, which was developed as a novel method for the accurate quantification of cfDNA. The patient died of septic shock on HD 8, suggesting that cfDNA could be used to monitor disease progression more effectively than PCT and the other inflammatory factors measured in this case.
CONCLUSION CfDNA may be a promising marker that complements other inflammatory factors to monitor disease progression in patients with septic shock.
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Affiliation(s)
- Jing-Ping Liu
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Shi-Chang Zhang
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Shi-Yang Pan
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
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Progress in quantitative technique of circulating cell free DNA and its role in cancer diagnosis and prognosis. Cancer Genet 2019; 239:75-84. [PMID: 31639530 DOI: 10.1016/j.cancergen.2019.10.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2019] [Revised: 07/24/2019] [Accepted: 10/09/2019] [Indexed: 01/21/2023]
Abstract
The interest in the potential application value of circulating cell free DNA (ccfDNA) has increased rapidly in recent years, as numerous researchers have demonstrated that the change of its level in the blood is associated with many diseases. Its potential role in cancer management is of particular concern. In comparison with traditional invasive tissue biopsy, quantitative analysis of ccfDNA level for the detection of cancer is advantageous due to the non-invasiveness of blood collection. Moreover, its clinical significance in prognosis prediction and dynamic monitoring of disease progression in cancer patients is equally worthy of attention. At the same time, quantitative detection of ccfDNA is being improved to pursue higher sensitivity due to its low concentration in the blood sample. In this review, we will summarize the progress in quantitative technology of ccfDNA and describe the possible relationship between ccfDNA level and cancer diagnosis and prognosis prediction.
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Xia WY, Gao L, Dai EH, Chen D, Xie EF, Yang L, Zhang SC, Zhang BF, Xu J, Pan SY. Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients. World J Gastroenterol 2019; 25:3985-3995. [PMID: 31413532 PMCID: PMC6689808 DOI: 10.3748/wjg.v25.i29.3985] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 06/13/2019] [Accepted: 07/05/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed.
AIM To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method.
METHODS A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay.
RESULTS Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P < 0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with marked-severe inflammation were significantly higher than those with mild-moderate inflammation (P < 0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R2 = 0.673, P < 0.01) or plasma DNA (R2 = 0.597, P < 0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild–moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively.
CONCLUSION The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.
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Affiliation(s)
- Wen-Ying Xia
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Li Gao
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Er-Hei Dai
- Department of Laboratory Medicine, the Fifth Hospital of Shijiazhuang, Shijiazhuang 050021, Hebei Province, China
| | - Dan Chen
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Er-Fu Xie
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Li Yang
- Department of Laboratory Medicine, the Fifth Hospital of Shijiazhuang, Shijiazhuang 050021, Hebei Province, China
| | - Shi-Chang Zhang
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Bing-Feng Zhang
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Jian Xu
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
| | - Shi-Yang Pan
- Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
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Gorgannezhad L, Umer M, Islam MN, Nguyen NT, Shiddiky MJA. Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies. LAB ON A CHIP 2018; 18:1174-1196. [PMID: 29569666 DOI: 10.1039/c8lc00100f] [Citation(s) in RCA: 195] [Impact Index Per Article: 27.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Cell-free DNA (cfDNA) refers to short fragments of acellular nucleic acids detectable in almost all body fluids, including blood, and is involved in various physiological and pathological phenomena such as immunity, coagulation, aging, and cancer. In cancer patients, a fraction of hematogenous cfDNA originates from tumors, termed circulating tumor DNA (ctDNA), and may carry the same mutations and genetic alterations as those of a primary tumor. Thus, ctDNA potentially provides an opportunity for noninvasive assessment of cancer. Recent advances in ctDNA analysis methods will potentially lead to the development of a liquid biopsy tool for the diagnosis, prognosis, therapy response monitoring, and tracking the rise of new mutant sub-clones in cancer patients. Over the past few decades, cancer-specific mutations in ctDNA have been detected using a variety of untargeted methods such as digital karyotyping, personalized analysis of rearranged ends (PARE), whole-genome sequencing of ctDNA, and targeted approaches such as conventional and digital PCR-based methods and deep sequencing-based technologies. More recently, several chip-based electrochemical sensors have been developed for the analysis of ctDNA in patient samples. This paper aims to comprehensively review the diagnostic, prognostic, and predictive potential of ctDNA as a minimally invasive liquid biopsy for cancer patients. We also present an overview of current advances in the analytical sensitivity and accuracy of ctDNA analysis methods as well as biological and technical challenges, which need to be resolved for the integration of ctDNA analysis into routine clinical practice.
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Affiliation(s)
- Lena Gorgannezhad
- School of Environment and Science, Griffith University, Nathan Campus, QLD 4111, Australia. and Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia
| | - Muhammad Umer
- Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia
| | - Md Nazmul Islam
- School of Environment and Science, Griffith University, Nathan Campus, QLD 4111, Australia. and Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia
| | - Nam-Trung Nguyen
- Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia
| | - Muhammad J A Shiddiky
- School of Environment and Science, Griffith University, Nathan Campus, QLD 4111, Australia. and Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia
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