All-in-One Fusogenic Nanoreactor for the Rapid Detection of Exosomal MicroRNAs for Breast Cancer Diagnosis.

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

Microbead-Based Colorimetric and Portable Sensors for Polyphenol Detection.

Natural polyphenols found in health supplements and drinks have antioxidant and anti-inflammatory properties. In particular, to determine the beneficial qualities of antioxidant drinks and beverages, consumers demand precise quantification of the total amount of polyphenols as on-site detection. Herein, we developed a new concept of portable beads suitable for the field detection available: colorimetric quantification of polyphenols equipped with color converting software applications in a smartphone or tablet PC. The yellowish beads contain ferric ions to react with polyphenol to produce blackish metal-phenolic complexes. It is simple to perform the detection procedure: dipping the beads in the analytical sample and out-taking a photo-converting into RGB color values and quantification of the existed polyphenol. The overall process was completed within 5 min. Compared with the Folin-Ciocalteu assay, which is a representative optical sensor kit for total phenolic content, the bead-based sensor showed a better limit of detection of 0.0415 mM for tannic acid and comparable sensing capability for a polyphenol-containing plant extract and brewed tea. The beads conserved the shape and sensitivity after months of storage or under environmental interference such as a change in the temperature.

Nucleic Acid Amplification Circuit-Based Hydrogel (NACH) Assay for One-Step Detection of Metastatic Gastric Cancer-Derived Exosomal miRNA.

Gastric cancer (GC) is recognized as the fifth most prevalent malignant tumor worldwide. It is characterized by diverse clinical symptoms, treatment responses, and prognoses. In GC prognosis, the promotion of epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, thereby triggering the dissemination of tumor cells. This study proposes a nucleic acid amplification circuit-based hydrogel (NACH) assay for identifying exosomal miRNA derived from metastatic GC. The NACH assay employs the rolling circle amplification method and targets miRNA-21, a tumor-related oncogene, and miRNA-99a, which promotes EMT. Specific amplification probes for each target are immobilized within the hydrogel, enabling a streamlined, one-step amplification reaction. The NACH assay exhibits a detection limit of 1 fm for miRNA-21 and miRNA-99a, thereby enabling rapid and highly sensitive on-site detection. Performance evaluation using exosomal miRNA extracted from cell culture media, mouse plasma, and human plasma revealed fluorescence intensity patterns similar to those obtained in qRT-PCR. Furthermore, deploying a custom-developed portable fluorometer for the NACH assay allows for diagnostic performance assessment and point-of-care testing using clinical samples from GC patients. These findings emphasize the potential of the NACH assay to be used as a robust tool for the genetic diagnosis of GC based on exosome detection.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Deep learning-assisted monitoring of trastuzumab efficacy in HER2-Overexpressing breast cancer via SERS immunoassays of tumor-derived urinary exosomal biomarkers.

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues.

On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR).

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

Multiplex Detection of Foodborne Pathogens using 3D Nanostructure Swab and Deep Learning-Based Classification of Raman Spectra.

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry.

Colorimetric Detection of HER2-Overexpressing-Cancer-Derived Exosomes in Mouse Urine Using Magnetic-Polydiacetylene Nanoparticles.

Breast cancer (BC) is a major global health problem, with ≈20-25% of patients overexpressing human epidermal growth factor receptor 2 (HER2), an aggressive marker, yet access to early detection and treatment varies across countries. A low-cost, equipment-free, and easy-to-use polydiacetylene (PDA)-based colorimetric sensor is developed for HER2-overexpressing cancer detection, designed for use in low- and middle-income countries (LMICs). PDA nanoparticles are first prepared through thin-film hydration. Subsequently, hydrophilic magnetic nanoparticles and HER2 antibodies are sequentially conjugated to them. The synthesized HER2-MPDA can be concentrated and separated by a magnetic field while inheriting the optical characteristics of PDA. The specific binding of HER2 antibody in HER2-MPDA to HER2 receptor in HER2-overexpressing exosomes causes a blue-to-red color change by altering the molecular structure of the PDA backbone. This colorimetric sensor can simultaneously separate and detect HER2-overexpressing exosomes. HER2-MPDA can detect HER2-overexpressing exosomes in the culture medium of HER2-overexpressing BC cells and in mouse urine samples from a HER2-overexpressing BC mouse model. It can selectively isolate and detect only HER2-overexpressing exosomes through magnetic separation, and its detection limit is found to be 8.5 × 108 particles mL-1. This colorimetric sensor can be used for point-of-care diagnosis of HER2-overexpressing BC in LMICs.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria.

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

Rapid and simultaneous multiple detection of a tripledemic using a dual-gate oxide semiconductor thin-film transistor-based immunosensor.

The simultaneous infection with a tripledemic-simultaneous infection with influenza A pH1N1 virus (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV)-necessitates the development of accurate and fast multiplex diagnostic tests. The coronavirus disease 2019 (COVID-19) pandemic has emphasized the importance of virus detection. Field-effect transistor (FET)-based immuno-biosensors have a short detection time and do not require labeling or polymerase chain reaction. This study demonstrates the rapid, sensitive detection of influenza A pH1N1, SARS-CoV-2, and RSV using a multiplex immunosensor based on a dual-gate oxide semiconductor thin-film transistor (TFT), a type of FET. The dual-gate oxide TFT was modified by adjusting both top and bottom gate insulators to improve capacitive coupling to approximately 120-fold amplification, exhibiting a high pH sensitivity of about 10 V/pH. The dual-gate oxide TFT-based immunosensor detected the target proteins (hemagglutinin (HA) protein of Flu, spike 1 (S1) protein of SARS-CoV-2, and fusion protein of RSV) of each virus, with a limit of detection of approximately 1 fg/mL. Cultured viruses in phosphate-buffered saline or artificial saliva and clinical nasopharynx samples were detected in 1-µL sample volumes within 60 s. This promising diagnosis could be potentially as point-of-care tests to facilitate a prompt response to future pandemics with high sensitivity and multiplexed detection without pretreatment.

A portable smartphone-based colorimetric sensor that utilizes dual amplification for the on-site detection of airborne bacteria.

Over the past few years, infections caused by airborne pathogens have spread worldwide, infecting several people and becoming an increasingly severe threat to public health. Therefore, there is an urgent need for developing airborne pathogen monitoring technology for use in confined environments to enable epidemic prevention. In this study, we designed a colorimetry-based bacterial detection platform that uses a clustered regularly interspaced short palindromic repeat-associated protein 12a system to amplify signals and a urease enzyme to induce color changes. Furthermore, we have developed a smartphone application that can distinguish colors under different illumination conditions based on the HSV model and detect three types of disease-causing bacteria. Even synthetic oligomers of a few picomoles of concentration and genomic DNA of airborne bacteria smaller than several nanograms can be detected with the naked eye and using color analysis systems. Furthermore, in the air capture model system, the bacterial sample generated approximately a 2-fold signal difference compared with that in the control group. This colorimetric detection method can be widely applied for public safety because it is easy to use and does not require complex equipment.

An immuno-magnetophoresis-based microfluidic chip to isolate and detect HER2-Positive cancer-derived exosomes via multiple separation.

Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer-derived exosomes. Therefore, a microfluidic chip-based magnetically labeled exosome isolation system (MEIS-chip) was developed as a lab-on-a-chip platform for human epidermal growth factor receptor 2 (HER2)-positive cancer diagnosis and monitoring. Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2-overexpressing and common exosomes using immunoaffinity. MNC-bonded exosomes were separated into different exits according to their magnetization degrees. The MEIS-chip efficiently separated HER2-overexpressing exosomes from common exosomes that did not contain disease-related information. The simultaneous separation of HER2-and non-HER2-overexpressing exosomes provided a means of analyzing high-purity HER2-overexpressing exosomes while minimizing the contribution of non-target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real-time changes in HER2 expression. These findings support the application of MEIS-chip for cancer diagnosis and treatment monitoring via effective exosome isolation.

Charge-shifting polyplex as a viral RNA extraction carrier for streamlined detection of infectious viruses.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, user-friendly nucleic acid testing that involves simple but efficient RNA extraction. Here, we present a charge-shifting polyplex as an RNA extraction carrier for advanced diagnosis of infectious viral diseases. The polyplex comprises poly(2-(dimethylamino) ethyl acrylate) (pDMAEA) electrostatically conjugated with RNA. The pDMAEA film can rapidly dissolve in the viral RNA solution, promoting immediate binding with RNA to form the polyplex, which enables the efficient capture of a substantial quantity of RNA. Subsequently, the captured RNA can be readily released by the quick hydrolysis of pDMAEA at the onset of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), streamlining the entire process from RNA extraction to analysis. The developed method requires only 5 min of centrifugation and enables the detection of RNA in a one-pot setup. Moreover, the proposed method is fully compatible with high-speed qRT-PCR kits and can identify clinical samples within 1 h including the entire extraction to detection procedure. Indeed, the method successfully detected influenza viruses, SARS-CoV-2, and their delta and omicron variants in 260 clinical samples with a sensitivity of 99.4% and specificity of 98.9%. This rapid, user-friendly polyplex-based approach represents a significant breakthrough in molecular diagnostics.

Conductive Thread-Based Immunosensor for Pandemic Influenza A (H1N1) Virus Detection.

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.

Polydiacetylene-based hydrogel beads as colorimetric sensors for the detection of biogenic amines in spoiled meat.

Ingesting large quantities of biogenic amines (BAs), which are released from spoiled foods, can have adverse side effects on the human body. Herein, we developed a colorimetric sensor using polydiacetylene (PDA)-based hydrogel beads that change color upon binding with BAs, thereby conveniently checking whether food is spoiled due to improper storage and distribution. The colorimetric sensor is fabricated by mixing PDA liposomes with an alginate solution. PDA undergoes a color change from blue to red when exposed to various external stimuli. In addition, alginate bestows the hydrogel with a three-dimensional porous structure, affording a large surface area. The PDA-based hydrogel beads can visually confirm the presence of BAs in solution or vapor form. Cadaverine and propylamine were rapidly detected with distinct color changes in the solution and vapor phases, respectively. The spoilage of pork meat at room temperature could be detected after two days as a 40.84% red chromatic shift.

A highly specific and flexible detection assay using collaborated actions of DNA-processing enzymes for identifying multiple gene expression signatures in breast cancer.

Most nucleic acid biosensors employ nucleic acid-processing enzymes to bind, degrade, splice, synthesize, and modify nucleic acids. Utilizing their unique substrate preference, binding mode, and catalytic activity is of great importance in designing nucleic acid biosensors. Combination with DNA-processing enzymes enables them to transform into a new generation of molecular diagnostics tools with enhanced selectivity and sensitivity and reduced reaction time. Here, we report an isothermal amplification strategy by coemploying a structure-specific endonuclease (flap endonuclease 1, FEN1) and a strand-displacing DNA polymerase (Bst DNA polymerase) to detect long RNA targets. This approach couples the FEN1-driven invasive cleavage reaction with toehold-mediated rolling circle amplification (iFEN-tRCA), enabling the highly selective and rapid detection of long RNA targets and offering a detection limit below 10 pM within 1 h. We used two targets, such as human epidermal growth factor receptor 2 (HER2, encoded by ERBB2) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP, encoded by PPP1R1B), associated with prognosis or response to anticancer therapy. We demonstrated the feasibility and quantitative capability of the iFEN-tRCA assay by assessing the expression of two RNA transcripts (ERBB2 and PPP1R1B) with total RNA extracts purified from human breast cancer cells. Therefore, we envision that the developed assay will provide a suitable prognostic and diagnostic tool for identifying appropriate patients for HER2-targeted therapy and predicting the clinical outcome and occurrence of metastasis relapse in breast cancer.

Identification and validation of methylated PENK gene for early detection of bladder cancer using urine DNA.

BACKGROUND: Early detection of bladder cancer (BCa) offers patients a favorable outcome and avoids the need for cystectomy. Development of an accurate and sensitive noninvasive BCa diagnostic test is imperative. DNA methylation is an early epigenetic event in the development of BCa. Certain specific aberrant methylations could serve as useful biomarkers. The aim of this study was to identify methylation biomarkers for early detection of BCa. METHODS: CpG methylation microarray analysis was conducted on primary tumors with varying stages (T1-T4) and paired nontumor tissues from nine BCa patients. Bisulfite-pyrosequencing was performed to confirm the methylation status of candidate genes in tissues and urine sediments (n = 51). Among them, PENK was selected as a potential candidate and validated using an independent set of 169 urine sediments (55 BCa, 25 benign urologic diseases, 8 other urologic cancers, and 81 healthy controls) with a quantitative methylation-specific real time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0. RESULTS: CpG methylation microarray analysis and stepwise validation by bisulfite-pyrosequencing for tissues and urine sediments supported aberrant methylation sites of the PENK gene as potential biomarkers for early detection of BCa. Clinical validation of the mePENK-qMSP test using urine sediment-DNA showed a sensitivity of 86.5% (95% CI: 71.2 - 95.5%), a specificity of 92.5% (95% CI: 85.7 - 96.7%), and an area under ROC of 0.920 (95% CI: 0.863 - 0.959) in detecting Ta high-grade and advanced tumor stages (T1-T4) of BCa patients. Sensitivities for Ta low-grade, Ta high-grade, T1 and T2-T4 were 55.6, 83.3, 88.5, and 100%, respectively. Methylation status of PENK was not correlated with sex, age or stage, while it was associated with the tumor grade of BCa. CONCLUSIONS: In this study, we analyzed the comprehensive patterns of DNA methylation identified that PENK methylation possesses a high potential as a biomarker for urine-based early detection of BCa. Validation of PENK methylation confirms that it could significantly improve the noninvasive detection of BCa.