Sensitive Detection Of DNA Methylation Research Articles

A ratiometric fluorescence assay for the detection of DNA methylation based on an alkaline phosphatase triggered in situ fluorogenic reaction.

The accurate and sensitive quantification of DNA methylation is significant for the early diagnosis of cancer. In this work, an alkaline phosphatase (ALP) triggered in situ fluorogenic reaction between ascorbic acid (AA) and 2,3-DAN was employed as a ratiometric fluorescent probe for the accurate and sensitive detection of DNA methylation with the assistance of ALP encapsulated liposomes. The quinoxaline derivative with a yellow fluorescence emission (I525) was generated from the reaction between AA and 2,3-DAN. Meanwhile, the consumption of 2,3-DAN declined its fluorescence intensity (I386). A ratiometric fluorescent probe (I525/I386) constructed by the above in situ fluorogenic reaction was applied for the accurate detection of DNA methylation. The methylated DNA was first captured by its complementary DNA in 96-well plates. Then, 5mC antibody (Ab) linked liposomes that were encapsulated with ALP recognized and combined with the methylation sites of the target DNA. After the liposomes were lysed by Triton X-100, the released ALP triggered the hydrolysis of ascorbic acid diphosphate (AAP) to form AA, participating in the fluorogenic reaction with 2,3-DAN to produce a quinoxaline derivative. Thus, the ratiometric fluorescence detection of DNA methylation was achieved using I525/I386 values. Using the ALP-enzyme catalyzed reaction and liposomes as signal amplifiers, a low detection limit of 82 fM was obtained for DNA methylation detection. Moreover, the accuracy of the assay could be improved using ratiometric fluorescent probes. We hope that the proposed assay will pave a new way for the accurate determination of low-abundance biomarkers.

GlaI cleavage assistant isothermal exponential amplification coupling with CRISPR/Cas12a for ultrasensitive detection of CLDN11 methylation: A potential marker for lung adenocarcinoma

DNA methylation is closely associated with cancer, and sensitive detection of DNA methylation is immensely significant for early cancer screening. In this study, for the first time, we identified CLDN11 methylation as a valuable marker for lung adenocarcinoma diagnosis and prognosis based on database analysis and experimental verification using clinical tissues and sera. Then, a flexible platform was proposed for ultrasensitive detection of CLDN11 methylation by integrating methylation-dependent restriction endonuclease GlaI cleavage with exponential amplification reaction (EXPAR) and CRISPR/Cas12a (GlaI–EXPAR–Cas12a platform). GlaI only recognizes and cleaves methylated target sites with excellent selectivity; the released fragments are primers for triggering EXPAR–CRISPR/Cas12a. Using Cas12a, false positives generated by EXPAR can be weakened to a negligible level. The proposed platform for detecting CLDN11 methylation achieved excellent performance, with a low limit of detection (1.25 × 10–15 M), and reliably identified CLDN11 methylation at an abundance of 0.1 % even in the presence of a large number of unmethylated fragments. More importantly, the proposed platform can be used for the quantification of human serum and genomic DNA, as well as to distinguish normal cells (HEK293) from cancer cells (H1299, A549 and H358). Through this integration platform, all 20 lung adenocarcinoma tissues exhibited significantly higher levels of CLDN11 methylation than a total of 16 paracancerous tissues. Taken together, the GlaI–EXPAR–Cas12a platform is ultrasensitive and convenient, and has perfect specificity. Further, it may function as a flexible platform for early screening of lung adenocarcinoma.

Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation.

DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5'-phosphate terminuses. After treating with Lambda, the sequence with 5'-phosphate modification will be degraded, leaving the single-stranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.

Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification.

Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation from clinical samples remains a challenge. Herein, we propose an easy-to-operate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system Assisted Methylation (CAM) approach for the sensitive detection of DNA methylation through the integration of rolling circle amplification and CRISPR-Cas12a-assisted cascade amplification. Briefly, bisulfite was employed to prepare the clinical samples so that the methylated DNA sequences trigger the subsequent triple signal amplifications, whilst the normal counterparts do not. The triple signal amplification procedure consists of methylated DNA sequence-based rolling circle amplification for a preliminary signal enhancement, a nicking enzyme-initiated target cleavage for a secondary amplification, and CRISPR-Cas12a enzyme-mediated trans-cleavage for a tertiary signal enhancement. This proposed approach reveals high sensitivity, which can even distinguish as low as 0.01% methylation levels from mixtures, paving the way towards the acceleration of methylation-based cancer diagnostics and management.

Reliable recognition of DNA methylation using bioanalysis of hybridization on the surface of Ag/GQD nanocomposite stabilized on poly (β-cyclodextrin): A new platform for DNA damage studies using genosensor technology.

Due to the role of DNA methylation in causing cancer in the present study, an innovative and inexpensive method was designed for the sensitive detection of DNA methylation. The silver-graphene quantum dots (Ag/GQDs) nano ink with high electrical conductivity was used as a substrate for genosensor fabrication toward identification of DNA hybridization. Also, poly (β-cyclodextrin) (p[β-CD]) has been used as a biointerface for the stabilization of Ag/GQD nano ink. The thiolated pDNA strand (5'-SH-TCCGCTTCCCGACCCGCACTCCGC-3') (as bioreceptor element) was fixed on the substrate and hybridized with methylated (5'-GC(M)GGAGTGC(M)GGGTC(M)GGGAAGC(M)GGA-3') and unmethylated (5'-GCGGAGTGCGGGTCGGGAAGCGGA-3') cDNAs, as target sequences were studied using electroanalysis methods. Under optimal conditions and using electrochemical techniques, the linear range was 1 am to 1 pm with LLOQ of 1aM. Finally, the designed DNA genosensor was used for detection of DNA methylation in human plasma samples and can be used to detect methylation in patient samples. It is expected that the designed DNA-based biodevice will be used to early stage diagnosis of cancer using monitoring of DNA methylation. Also, this type of genosensor can be used for epigenetic studies in the near future.

Ultrasensitive DNA hypermethylation detection using plasma for early detection of NSCLC: a study in Chinese patients with very small nodules

PurposeWe had previously developed highly sensitive DNA methylation detection to diagnose lung cancer in patients with pulmonary nodules. To validate this approach and determine clinical utility in Chinese patients with indeterminate pulmonary nodules, we assessed the diagnostic accuracy for early stage lung cancer in plasma samples.Experimental designPatients with CT-detected small lung nodules (diameter ≤ 3.0 cm) were included. Cases (n = 163) had staged IA or IB non-small cell lung cancer (NSCLC), while controls (n = 83) had non-cancerous lesions. Promoter methylation of eight lung cancer-specific genes (CDO1, TAC1, SOX17, HOXA7, HOXA9, GATA4, GATA5, and PAX5) was detected using nanoparticle-based DNA extraction (MOB) followed by qMSP.ResultsMethylation detection for CDO1, TAC1, SOX17, and HOXA7 in plasma was significantly higher in cases compared with the benign group (p < 0.001). The sensitivity and specificity for lung cancer diagnosis using individual gene was 41–69% and 49–82%. A three-gene combination of the best individual genes has sensitivity and specificity of 90% and 71%, with area under the receiver operating curve (AUC) of 0.88, (95% CI 0.84–0.93). Furthermore, three-gene combinations detected even the smallest lung nodules, with the combination of CDO1, SOX17, and HOXA7 having the overall best performance, while the combination of CDO1, TAC1, and SOX17 was best in tumor sizes less than 1.0 cm.ConclusionsUsing modified MOB-qMSP, high sensitivity and specificity, for the detection of circulating tumor DNA was obtained for early stage NSCLC. This strategy has great potential to identify patients at high risk and improve the diagnosis of lung cancer at an earlier stage.Graphical

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies.

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

A simple and universal electrochemical assay for sensitive detection of DNA methylation, methyltransferase activity and screening of inhibitors

Many studies have confirmed that DNA methylation is highly correlated with the occurrence and development of various diseases including cancers. In this work, we developed a simple, sensitive, selective, and universal electrochemical biosensor for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using M.SssI MTase as an example. The thiolated single-stranded DNA S1 was self-assembled on the surface of gold nanoparticles deposition modified glassy carbon electrode via AuS bonding, then hybridization between the DNA S1 and its complementary DNA S2 formed a double-stranded target sequence for both M.SssI MTase and restriction endonuclease HpaII. HpaII could not cleave the target sequence after it was methylated by M.SssI MTase, while the sequence without methylation could be cleaved. Here, we used methylene blue (MB) as electrochemical indicator. The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL. Moreover, screening of M.SssI MTase inhibitors 5-azacytidine (5-Aza) and 5-Aza-2′-deoxycytidine (5-Aza-dC) were successfully investigated using the fabricated electrochemical biosensor and showed that the two classic drugs could both inhibit the M.SssI MTase activity with the IC50 of 2.8 μM and 0.37 μM, respectively, indicating potential application in discovery of new anticancer drugs.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites.

DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X' and Y') for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X' and Y' to generate the X'Y' products. The resultant X'Y' products may in turn act as the templates to ligate probes X and Y for the generation of XY products, consequently inducing tricyclic LCR amplification under thermal denaturation conditions to generate a large number of XY products. The subsequent hybridization of XY products with the capture and reporter probes results in the formation of sandwich hybrids which may assemble on the 605QD surface to obtain 605QD-oligonucleotide-Cy5 nanostructures, inducing efficient FRET from the 605QD to Cy5 and the emission of Cy5. This nanosensor can detect DNA methylation at single 5-methylcytosine (5-mC) resolution with a detection limit of as low as 1.0 aM and a large dynamic range of 7 orders of magnitude. Moreover, this nanosensor can distinguish as low as a 0.01% methylation level, and it can detect DNA methylation in human lung cancer cells as well, holding great potential for accurate epigenetic evaluation and early cancer diagnosis.

Label-free electrochemical detection of methyltransferase activity and inhibitor screening based on endonuclease HpaII and the deposition of polyaniline

Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III, as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5-0.6 U mL(-1) with the detection limit of 0.12 U mL(-1). G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.

Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase (MTase) activity was developed by using the methylation-sensitive restriction endonuclease Dpn I and methylation-responsive hairpin-capture DNA probe to improve selectivity and by employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The ECL biosensing electrode was fabricated by self-assembling a design of 5′-thiol modified methylation-responsive hairpin-capture DNA probe on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosylmethionine were introduced, all adenines in the symmetric tetranucleotide 5′-GATC-3′ of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by Dpn I endonuclease, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the “capture DNA probe” section on the gold electrode. Subsequently, the remained “capture DNA probe” on the gold electrode can hybridize with the 5′-amino modified DNA probe. Finally, tris(1, 10-phenanthroline) ruthenium (Ru(phen)32+)-assembled GO composites were conjugated to the electrode surface via EDC–NHS coupling, a strong ECL response was electrochemically generated. The increased ECL intensity was proportion to Dam MTase activity in the range from 0.05U/mL to 40U/mL with a detection limit of 0.02U/mL. The present work demonstrates that the combination of the enzyme-linkage and GO as a platform for signal probe is a great promising approach for MTase activity and evaluation of the capability of inhibitors for the MTase.

DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening

A sensitive and selective electrochemical method was developed for the detection of DNA methylation, determination of DNA methyltransferase (MTase) activity and screening of MTase inhibitor. Methylene blue (MB) was employed as electrochemical indicator and DNA-modified gold nanoparticles (AuNPs) were used as signal amplification unit because the DNA strands in this composite have strong adsorption ability for MB. First, the thiolated single-stranded DNA S1 was self-assembled on gold electrode, hybridization between the lower portion of DNA S1 and its complementary DNA S2 formed an identical double-stranded tetranucleotide target sequence for both DNA adenine methylation (Dam) MTase and methylation-resistant endonuclease Mbo I, then the upper portion of DNA S1 was hybridized with its complementary DNA S3 modified on AuNPs to bring the DNA S3-AuNPs amplification units onto the electrode. The DNA S1/S2/S3-AuNPs bioconjugate has lots of DNA strands, and they can adsorb abundant MB. Mbo I endounuclease could not cleave the identical target sequence after it was methylated by Dam MTase. On the contrary, the sequence without methylation could be cleaved, which would decrease the amount of adsorbed MB. The presence of redox-active MB was detected electrochemically by differential pulse voltammetry (DPV). Thus, the activity of Dam MTase and methylation status were sensitively converted to the DNA S3-AuNPs amplified DPV signals. The DPV signal demonstrated a linear relationship with logarithm of Dam concentration ranging from 0.075 to 30U/mL, achieving a detection limit of 0.02U/mL (S/N=3). Also, screening of Dam MTase inhibitor 5-fluorouracil was successfully investigated using this fabricated sensor.

Nanoparticle-enabled highly sensitive detection of DNA methylation in single tube

Abstract Background: One of the often occurred epigenetic events associated to cancer is inheritable transcriptional silencing of the tumor suppressor genes by aberrant methylation of cytosines at the promoter regions. Current approaches to detect promoter DNA methylation commonly involve three separate independent processes, including DNA extraction, bisulfite conversion and methylation detection through PCR amplification, such as methylation specific PCR (MSP). This method includes many disconnected steps with loss of genetic materials, potentially reducing the sensitivity required for analysis of challenging clinical samples. Methods and Results: To address this problem, we developed a new technique named Methylation-on-Beads (MOB). MOB uses the silica superparamagnetic nanobeads (SSB) to combine DNA extraction, bisulfite treatment (Bst) and PCR into a single tube. The single-tube scheme minimizes the DNA loss during the tube transfers and improves the analytical sensitivity for methylation detection. Further enhancement in sensitivity is achieved by combining MOB with quantum dot-enhanced MSP detection. The pre-PCR DNA yields using MOB were compared with those carried out with conventional organic solvent extraction and ethanol precipitation (PC). 15 serum samples from lung cancer patients (7 Stage I, 3 Stage II, 5 Stage III) were analyzed using both MOB and PC. The DNA recovery using MOB was higher than PC for each patient serum sample with a median increase of 6.61 fold. To demonstrate the versatile applicability of the new method, we further analyzed 10 samples to include fresh tissue and paraffin embedded tissue from normal patients, fresh tumors from cancer patients and sputum samples. Median DNA yield increase of 7.8, 5.3, 6.4 and 7.5 respectively was determined using MOB when compared to the conventional method. We examined the outcome of bisulfite conversion in the presence of SSBs using real-time methylation specific PCR (MSP) to analyze p16INK4a promoter methylation. A set of triplicate reactions were examined with bisulfite-treated DNA of varying treatment durations (0h, 1h, 3h, 4h, 8h), and compared to the control using 16 hrs of conventional bisulfite treatment. Results indicated that 4h of bisulfite treatment was sufficient for conversion, and that the presence of beads did not alter the conversion process. Assessment of methylation in serum or plasma can be a useful tool for early detection of cancer. However, extending the DNA methylation analysis to clinically usable serum/ blood-based tests has been limited by the lack of sensitivity of conventional methods. In order to address whether improving DNA yields could enhance methylation detection, we compared methylation of p16INK4a promoter in 49 patient serum samples (18 normal and 31 cancer) in a blinded study using both MOB and PC/Bst/MSP. The 31 tumor samples were pre-selected from patients diagnosed with lung cancer who were also methylated for p16INK4a promoter in corresponding tumors. While p16INK4a methylation was detected in 14/31 patients with lung cancer using conventional approach; using MOB, we were able to detect p16INK4a methylation in 23/31of these patients. When samples used for methylation analysis contained large amounts of DNA (cell lines, tumors etc), a single-tube analysis of entire input amount may be unnecessary. Instead, multiple reactions in parallel were feasible by directly splitting the SSB into several different tubes. Conclusion: MOB successfully combined three processes required for DNA methylation analysis into a single-tube using SSB thereby allowing for ease in handling and increased throughput in detection. Increased pre-PCR yield in MOB allowed for efficient, diagnostically sensitive methylation detection.

Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection.

DNA methylation is an epigenetic modification that occurs at 5′ cytosine in CpG dinucleotides and is observed in high frequency in the transcription promoter regions of tumor-suppressor genes.[1,2] Such changes account for the loss of function of tumor-suppressor genes for almost every type of cancer;[3] and more genes are inactivated by promoter hyper-methylation than by genetic mutations.[4] These abnormal epigenetic changes appear to be an early event that precedes the occurrence of genetic mutations.[5-8] Hence, detecting DNA promoter methylation has significant clinical and prognostic implications for early cancer detection. Methylation-specific PCR (MSP) remains one of the most widely used methods for the sensitive detection of DNA methylation,[9] but requires gel electrophoresis and is limited to qualitative analysis. Fluorescence-based MSP variants[10,11] utilize fluorescent probes for real-time detection, thereby facilitating high-throughput and quantitative detection of DNA methylation. In recent years, quantum dots (QDs) have emerged as promising fluorophores for biological sensing due to their unique spectroscopic properties, such as broad excitation spectra, narrow size-tunable emission wavelengths, and high photostability. QDs have also been used as FRET donors to detect biomolecular targets.[12-15] The large Stokes shift and narrow emission spectra of QDs allow for FRET with a significantly reduced acceptor excitation and spectral crosstalk, and thus permits the design of QD-FRET biosensors with the minimal background necessary for detecting targets at low-concentration.[13,16] Recently, this feature of low intrinsic background of QD-FRET facilitated the development of a methylation-detection method that achieves greater sensitivity over conventional MSP methods, as exemplified by analyzing sputum samples from lung cancer patients.[16]

Sensitive detection of DNA methylation.

In recent years, many molecular biomarkers have been discovered that are capable of distinguishing tumors from normal tissue. Among the different types of markers, DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or diluted by excess normal DNA. One of the most widely used methods for measuring methylation levels, methylation-specific PCR (MSP), has been proved to be a very effective tool in situations requiring sensitive detection. The addition of fluorogenic probes makes these assays more informative, quantitative, and suitable for a clinical format. The field of sensitive detection is not limited to MSP; hence, an alternative methylation-sensitive amplification is discussed. PCR-based methylation assays have been applied to the detection of tumor DNA in a variety of body fluids, including serum, plasma, urine, sputum, and lavage fluids. In many cases, the sensitivity and specificity of these detection assays has been impressive, but important technological issues remain in areas such as sample preparation, assay design, and marker selection. Once these technical concerns have been addressed, the sensitive detection of methylation will provide a powerful diagnostic and prognostic tool, especially for the early detection of preneoplastic and neoplastic lesions.