A novel "on-off-on" electrochemiluminescence aptasensor based on resonance energy transfer as signal amplification strategy for ultrasensitive detection of sulfadiazine in different samples.

The misuse of antibiotics may contaminate the environment and cause harm to human health. Therefore, rapid and accurate detection of antibiotics is essential. In this study, a novel electrochemiluminescence resonance energy transfer (ECL-RET) pair was designed using a new ECL emitter (CPM, Ce-TBAPy) as the donor and Co-MOF@AuPt as the acceptor. Moreover, a highly sensitive and specific "on-off-on" ECL aptasensor was constructed for detecting sulfadiazine (SDZ). The aptasensor exhibited a broad linear range (from 10.0 fg mL-1 to 100 ng mL-1) for the SDZ concentration, with limit of detection and limit of quantification values of 1.14 fg mL-1and 3.75 fg mL-1, respectively. The aptasensor achieved good results in spiking experiments with milk and egg samples, and successfully quantified SDZ in fish meal quality control sample. The prepared aptasensor presents great potential for food and environmental safety by detecting antibiotics.

Ternary electrochemiluminescence quenching effects of CuFe2O4@PDA-MB towards self-enhanced Ru(dcbpy)32+ functionalized 2D metal-organic layer and application in carcinoembryonic antigen immunosensing.

BACKGROUND: Carcinoembryonic antigen (CEA) is a significant glycosylated protein, and the unusual expression of CEA in human serum is used as a tumor marker in the clinical diagnosis of many cancers. Although scientists have reported many ways to detect CEA in recent years, such as electrochemistry, photoelectrochemistry, and fluorescence, their operation is complex and sensitivity is average. Therefore, finding a convenient method to accurately detect CEA is significance for the prevention of malignant tumors. With high sensitivity, quick reaction, and low background, electrochemiluminescence (ECL) has emerged as an essential method for the detection of tumor markers in blood. RESULTS: In this work, a "signal on-off" ECL immunosensor for sensitive analysis of CEA ground on the ternary extinction effects of CuFe2O4@PDA-MB towards a self-enhanced Ru(dcbpy)32+ functionalized metal-organic layer [(Hf)MOL-Ru-PEI-Pd] was prepared. The high ECL efficiency of (Hf)MOL-Ru-PEI-Pd originated from the dual intramolecular self-catalysis, including intramolecular co-reaction between polyethylenimine (PEI) and Ru(dcbpy)32+. At the same time, loading Pd NPs onto (Hf)MOL-Ru-PEI could not only improve the electron transfer ability of (Hf)MOL-Ru-PEI, but also provide more active sites for the reaction of Ru(dcbpy)32+ and PEI. In the presence of CEA, CuFe2O4@PDA-MB-Ab2 efficiently quenches the excited states of (Hf)MOL-Ru-PEI-Pd by PDA, Cu2+, and methylene blue (MB) via energy and electron transfer, leading to an ECL signal decrease. Under optimal conditions, the proposed CEA sensing strategy showed satisfactory properties ranging from 0.1 pg mL-1 to 100 ng mL-1 with a detection limit of 20 fg mL-1. SIGNIFICANCE: The (Hf)MOL-Ru-PEI-Pd and CuFe2O4@PDA-MB were prepared in this work might open up innovative directions to synthesize luminescence-functionalized MOLs and effective quencher. Besides, the ECL quenching mechanism of Ru(dcbpy)32+ by MB was successfully explained by the inner filter effect (ECL-IFE). At last, the proposed immunosensor exhibits excellent repeatability, stability, and selectivity, and may provide an attractive way for CEA and other disease markers determination.

Wheel drive-based DNA sensing system for highly specific and rapid one-step detection of MiRNAs at the attomolar level.

With the use of DNA as building blocks, a variety of microRNA amplification-based sensing systems have been developed. Nevertheless, ultrasensitive, selective and rapid detection of microRNAs with a high signal-to-background ratio and point mutation discrimination ability remains a challenge. Herein, we propose a novel wheel drive-based DNA sensing system (NWDS) based on a self-assembled, self-quenched nanoprobe (SQP) to conduct highly specific and ultrasensitive one-step measurement of microRNAs. In this work, a signalling recognition DNA hairpin (DH) sequence with a self-complementary stem domain of 14 base pairs was used, which contained three functional regions, namely a recognition region for the target miRNA-21, a sticky region with 9 complementary nucleotides to the 3'terminus of a DNA wheel (DW) and a region for the hybridization with a quenching DNA primer (DP). The SQP was ingeniously self-assembled at room temperature by the DH and DP, which was capable of eliminating unwanted background signals. MiRNA-21 was employed as a target model to specifically activate the SQP, leading to specific hybridization between the HP and DW. With the assistance of a polymerase, an SQP-based wheel driving took place to induce hybridization/polymerization displacement cycles, initiating target recycling and DP displacement. As a result, a large amount of the newly formed hybrid SQP/DW accumulated to generate a substantially enhanced fluorescence signal. In this way, the newly proposed NWDS exhibits ultrasensitivity with a detection limit of 5.62 aM across a wide linear dynamic response range up to 200 nM, excellent selectivity with the capability to discriminate homologous miRNAs and one-base, two-base and three-base mismatched sequences, and an outstanding analytical performance in complex systems. In addition, the significant simultaneous advantages of one-step operation, rapid detection within 15 min and a high signal-to-background ratio of 26 offer a unique opportunity to promote the early diagnosis of cancer-related diseases and molecular biological analysis.

Evaluation of a new point-of-care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS: Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS: The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS: The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

Fluorescent quenching probes based SAA 1 genotyping with a fully automated system.

OBJECTIVE: The aim of the present study is to develop and validate a reliable and simple application for genotyping serum amyloid A1 (SAA1). METHODS: The specific nested PCR was performed to amplify a product of SAA1 gene. Two quenching probes (QPs) were designed for detecting two single nucleotide polymorphism (SNP) sites, rs1136743(C/T) and rs1136747(C/T) respectively for SAA1 genotypes. The specific nested PCR and QPs of SAA1 genotying was introduced into a fully automated genotyping system (I-densy, ARKRAY, Inc.), which enables the genotyping of SAA1 from whole blood. RESULTS: Six genotypes of SAA1 (α+/+, ß+/+, γ+/+, αß, αγ and ßγ) could be determined by monitoring the fluorescence intensity of two QPs with melting temperature (TM) analysis. Total 121 clinical samples were SAA1 genotyped in the fluorescent quenching probes based method with a fully automated I-densy system and were further sequence confirmed with a PCR direct sequencing approach. CONCLUSION: This fully automated system is a rapid and reliable strategy for the SAA1 genotyping and for its future clinical application.

Clinical Evaluation of a Novel Point-of-Care Assay To Detect Mycoplasma pneumoniae and Associated Macrolide-Resistant Mutations.

The recent increase in macrolide-resistant Mycoplasma pneumoniae in Asia has become a continuing problem. A point-of-care testing method that can quickly detect M. pneumoniae and macrolide-resistant mutations (MR mutations) is critical for proper antimicrobial use. Smart Gene (Mizuho Medy Co., Ltd., Tosu City, Saga, Japan) is a compact and inexpensive fully automatic gene analyzer that combines amplification with PCR and the quenching probe method to specify the gene and MR mutations simultaneously. We performed a clinical evaluation of this device and its reagents on pediatric patients with suspected M. pneumoniae respiratory infections and evaluated the impact of the assay on antimicrobial selection. Using real-time PCR as a comparison control, the sensitivity of Smart Gene was 97.8% (44/45), its specificity was 93.3% (98/105), and its overall concordance rate was 94.7% (142/150). The overall concordance rate of Smart Gene diagnosis of MR mutations in comparison with sequence analysis was 100% (48/48). The ratio of MR mutations was significantly higher at high-level medical institutions than at a primary medical clinic (P = 0.023), and changes in antibiotic therapy to drugs other than macrolides were significantly more common in patients with MR mutations (P = 0.00024). Smart Gene demonstrated excellent utility in the diagnosis of M. pneumoniae and the selection of appropriate antimicrobials for MR mutations at primary medical institutions, which play a central role in community-acquired pneumonia care. The use of this device may reduce referrals to high-level medical institutions for respiratory infections, thereby reducing the medical and economic burdens on patients.

Development and evaluation of a novel quenching probe PCR (GENECUBE) assay for rapidly detecting and distinguishing between Chlamydia pneumoniae and Chlamydia psittaci.

Early detection of the family Chlamydiaceae as pathogens is essential worldwide for the rapid and sufficient management of atypical pneumonia. GENECUBE (TOYOBO) is a novel fully automated gene analyzer capable of amplifying and detecting target DNAs within 50 min. In this study, we developed a new PCR assay with a specific quenching probe (PCR-QC assay) for rapidly distinguishing between Chlamydia pneumoniae (CPN) and Chlamydia psittaci (CPS). The PCR-QC assay enabled us to precisely and simultaneously detect the 2 different types of DNA fragments even in a mixed sample by identifying unique melting temperatures. Next, we examined a total of 300 frozen samples from patients with respiratory tract infection using the PCR-QC assay and the cell culture method as the gold standard. Kappa index for agreement between the PCR-QC assay and the culture method was 0.43 (95% confidential interval (CI): 0.08-0.78). The sensitivity and specificity of the PCR-QC assay were 36.3% (4/11; 95% CI: 10.9-69.2%)) and 99.0% (286/289; 95% CI: 97.0-99.8%), respectively. The samples positive for CPN (n = 13) or CPS (n = 1) by either method were also examined by a conventional PCR TaqMan assay, which produced the same results as those from the PCR-QC assay. Furthermore, the PCR-QC assay using GENECUBE shortened the full detection time for CPN or CPS (within 50 min vs. more than 2 to 3 h) compared with conventional PCR TaqMan assays. Therefore, the new PCR-QC assay system equipped with GENECUBE is useful for rapidly detecting CPN or CPS pathogens in clinical laboratory, and may improve the management of atypical pneumonia.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Evaluation of GENECUBE Mycoplasma for the detection of macrolide-resistant Mycoplasma pneumoniae.

Introduction. Resistance against macrolide antibiotics in Mycoplasma pneumoniae is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of Mycoplasma pneumoniae as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA).Hypothesis/Gap Statement. The automated molecular diagnostic sytem can identify macrolide-resistant M. pneumoniae.Aim. To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations.Methodology. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from M. pneumoniae-positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of M. pneumoniae positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed.Results. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed M. pneumoniae-positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing.Conclusion. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant M. pneumoniae.

High fluorescence quenching probe-based reverse fluorescence enhancement LFTS coupling with IS-primer amplification reaction for the rapid and sensitive Parkinson Disease-associated MicroRNA detection.

Parkinson Disease (PD) is the second-most common neurodegenerative disorder in the population. Recent researches indicated that hsa-microRNA 5010-3p (miR-5010) and hsa-microRNA 331-5p (miR-331) were significantly important for the detection of PD. So, in this work, a kind of high fluorescence quenching probe-based reverse fluorescence enhancement lateral flow test strip (rLFTS) was constructed to realize the synchronous detection of miR-5010 and miR-331. The formation of black hole quencher 2 (BHQ2) coating gold nanoparticles (AuNPs) effectively enhanced the fluorescence quenching property of the probes so as to significantly improve the detection sensitivity. This rLFTS also coupled with "invading stacking primer" (IS-primer) isothermal amplification reaction (ISAR) to accomplish rapid, sensitive, specific, and synchronous detection of PD-associated microRNA (miRNA). The whole detection time was shorter (35 min), and the limit-of-detection (LOD) reached to fM level. For the high accuracy diagnosis of PD, the synchronous determination of miR-5010 and miR-331 was successfully realized on one rLFTS by labeling fluorescent molecules to different T-line. This rLFTS also allowed for miRNA detection in total microRNA extracts from whole blood samples of PD patients, which performed important value in PD diagnosis and biomedical research.

Detection of the Genus Phytophthora and the Species Phytophthora nicotianae by LAMP with a QProbe.

Phytophthora is an oomycete genus with worldwide distribution, and many of its species cause destructive diseases. In Japan, Phytophthora species are listed as quarantine organisms with the exception of Phytophthora nicotianae. For effective quarantine control, we designed a Phytophthora genus-specific loop-mediated isothermal amplification (LAMP) primer set and a P. nicotianae species-specific quenching probe (QProbe) to establish a simultaneous LAMP-based detection method. We confirmed the specificity of the genus-specific primers, and all 161 taxa were detected. No other species in the closely related genera Pythium and Phytopythium gave positive results with the exception of two species, Phytopythium delawarense and Phytopythium fagopyri. These two species gave inconsistent results. We used annealing curve analysis with the QProbe to demonstrate that P. nicotianae could be distinguished from other species. DNA from inoculated and naturally infected plants was extracted using a time-saving extraction kit and subjected to the simultaneous detection method. We confirmed that all Phytophthora DNAs in the plant samples were detected, and P. nicotianae was specifically identified. This simultaneous detection method will make quarantine inspections faster and easier.

Identification of the BRAF V600E mutation in Japanese patients with hairy cell leukemia and related diseases using a quenching probe method.

Hairy cell leukemia (HCL) is a rare B-cell lymphoid malignancy that is difficult to distinguish from other morphological variants. The frequency of HCL has not been determined accurately in Japan. Recent studies revealed that the BRAF V600E mutation is the causal genetic event in HCL. We assessed the BRAF mutation in Japanese patients with HCL and related diseases using the quenching probe (QP) method, a single-nucleotide polymorphism detection system, and evaluated the incidence rate of HCL among Japanese patients with chronic lymphocytic leukemia, and related diseases. We identified 18 cases (33.3%) harboring the BRAF mutation among 54 patients diagnosed with, or suspected of having HCL. Of BRAF V600E-positive patients, 7 were only detected using the QP method, not by direct sequencing, whereas 11 were positive using both tests. In a larger cohort of Japanese patients diagnosed with chronic lymphoid leukemia or related diseases, the frequency of HCL was 4%. Patients with the BRAF V600E mutation had a significantly higher frequency of neutropenia, thrombocytopenia, and elevated soluble interleukin-2 receptor and common B-cell surface markers than patients without the mutation. Our results confirm that BRAF V600E-positive HCL is a relatively rare disorder in the Japanese leukemia patient population.

Amplified electrochemiluminescence detection of CEA based on magnetic Fe3O4@Au nanoparticles-assembled Ru@SiO2 nanocomposites combined with multiple cycling amplification strategy.

In this work, we designed a new strategy for ultrasensitive detection of CEA based on efficient electrochemiluminescence (ECL) quenching of Ru(bpy)32+-doped SiO2 nanocomposite by ferrocene using target recycling amplification technique. A large number of Ru@SiO2 ECL signal probe were firstly assembled on the novel magnetic core-shell Fe3O4@Au nanoparticles (NPs), then the ferrocene-labeled ECL quenching probe (Fc-probe) was linked to the magnetic NPs. Finally, numerous DNA1 sequences were produced by target CEA-triggered multiple recycling amplification and displaced the Fc-probe on the magnetic NPs, leading to significantly enhanced ECL signal for CEA detection. Because of the designed cascade signal amplification strategy, the newly developed method achieved a wide linear range of 10 fg/mL to 10 ng/mL with a low detection limit of  3.5 fg/mL. Furthermore, taking advantages of the magnetic Fe3O4@Au NPs for carring abundant signal probes, sensing target and ECL detection, the developed ECL strategy is convenient, rapid and displayed high sensitivity for CEA detection, which has great potential for analyzing the clinical samples in practical disease diagnosis applications.

PIK3CA mutation profiling in patients with breast cancer, using a highly sensitive detection system.

PIK3CA mutations are common activating mutations associated with breast cancer (occurring in 20-30% of all cases) and are potent predictive markers for responses to PI3K inhibitors. Thus, it is important to develop sensitive methods to detect these mutations. We established a novel detection method using a quenching probe (QP) system to identify PIK3CA mutations, using DNA from 309 breast cancer tissues. In a developmental cohort, we determined the optimal detection threshold of the QP system with human tumor DNA from 119 freshly frozen tumor samples. We found a 96% concordance rate with the QP system between DNA from 26 matching fresh-frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens from the same patients, and known PIK3CA mutation status in the developmental cohort. In a validation cohort, we evaluated whether the threshold for judging mutations using the QP system with frozen specimen-derived DNA was applicable with FFPE-derived DNA. In the validation cohort, 30 DNA samples from 190 FFPE-derived DNA samples with known PIK3CA mutation status were analyzed by direct sequencing (DS) and droplet digital PCR, in a blinded manner. The sensitivity and specificity of the droplet digital PCR results were 100% and 100% (QP system), and 60% and 100% (DS), respectively. We also analyzed the relationship between clinical outcomes and the PIK3CA mutational status of 309 breast cancer samples, including the developmental cohort and validation cohort samples. Multivariate analysis suggested that PIK3CA mutations, especially H1047R, were prognostic factors of relapse-free survival. Our novel detection system could be more useful than DS for detecting clinical PIK3CA mutations.

Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.

Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.

Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

INTRODUCTION: The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. METHOD: A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. RESULTS: For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. CONCLUSION: The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR.

Preparation and in vitro evaluation of methoxy polyethylene glycol-polycaprolactone nanoparticles loading a water-quenching fluorescent probe / 第二军医大学学报

Objective To prepare methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) nanoparticles loading a water-quenching fluorescent probe and to evaluate its in vitro characteristics and stability.Methods PCL nanoparticles and mPEG-PCL nanoparticles with different mPEG chain lengths (mPEG5k,mPEG2k) loading the waterquenching fluorescent probe P2 were prepared by an emulsification/solvent evaporation method using PCL and mPEG-PCL as materials.In vitro characteristics such as morphology,particle size and distribution were evaluated.The P2 probe displayed fluorescent signals when encapsulated in the matrix of the nanoparticles,but quenched immediately when released into water.Based on this properties of P2 probe,the stability of various nanoparticles in different aqueous media was investigated.Results The prepared mPEG-PCL nanoparticles showed a particle size of about 200 nm,narrow size distribution,polydispersity index below 0.06 and near neutral surface potentials with spherical morphology and smooth surfaces.In buffers of different pHs and simulated bio-relevant media,all nanoparticles showed very good stability without significant change in particle size,polydispersity index and fluorescence intensity.Conclusion mPEG-PCL nanoparticles have promising in vitro characteristics and robust stability.It is fast and convenient to monitor the stability of nanoparticles by using the water-quenching probe.

Preparation and in vitro evaluation of methoxy polyethylene glycol-polycaprolactone nanoparticles loading a water-quenching fluorescent probe / 第二军医大学学报

Objective To prepare methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) nanoparticles loading a water-quenching fluorescent probe and to evaluate its in vitro characteristics and stability. Methods PCL nanoparticles and mPEG-PCL nanoparticles with different mPEG chain lengths (mPEG5k, mPEG2k) loading the water-quenching fluorescent probe P2 were prepared by an emulsification/solvent evaporation method using PCL and mPEG-PCL as materials. In vitro characteristics such as morphology, particle size and distribution were evaluated. The P2 probe displayed fluorescent signals when encapsulated in the matrix of the nanoparticles, but quenched immediately when released into water. Based on this properties of P2 probe, the stability of various nanoparticles in different aqueous media was investigated. Results The prepared mPEG-PCL nanoparticles showed a particle size of about 200 nm, narrow size distribution, polydispersity index below 0.06 and near neutral surface potentials with spherical morphology and smooth surfaces. In buffers of different pHs and simulated bio-relevant media, all nanoparticles showed very good stability without significant change in particle size, polydispersity index and fluorescence intensity. Conclusion mPEG-PCL nanoparticles have promising in vitro characteristics and robust stability. It is fast and convenient to monitor the stability of nanoparticles by using the water-quenching probe.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.