Synthesis of Isolated DNA Aptamer and Its Application of AC-Electrothermal Flow-Based Rapid Biosensor for the Detection of Dengue Virus in a Spiked Sample.

Dengue fever is an infectious disease caused by the dengue virus (DENV) and is transmitted by mosquitoes in tropical and subtropical regions. The early detection method at a low cost is essential. To address this, we synthesized the isolated DENV aptamer for fabricating a rapid electrochemical biosensor on a Au interdigitated microgap electrode (AuIMGE). The DENV aptamers were generated using the SELEX (systemic evolution of ligands by exponential enrichment) method for binding to DENV surface envelope proteins. To reduce the manufacturing cost, unnecessary nucleotide sequences were excluded from the isolation process of the DENV aptamer. To reduce the detection time, the alternating current electrothermal flow (ACEF) technique was applied to the fabricated biosensor, which can shorten the detection time to 10 min. The performance of the biosensor was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In the diluted DENV protein solution, the linear range of the concentrations was from 1 pM to 1 µM and the LOD was 76.7 fM. Moreover, the proposed biosensor detected DENV in a diluted spiked sample at a linear range of 10-6 to 106 TCID50/mL, while the detection performance was proven with an LOD of 1.74 × 10-7 TCID50/mL along with high selectivity.

Fabrication of graphene oxide-based pretreatment filter and Electrochemical-CRISPR biosensor for the field-ready cyanobacteria monitoring system.

Microcystis aeruginosa (M. aeruginosa) cause the eutrophication of lakes and rivers. To effectively control the overgrowth of M. aeruginosa, a suitable measurement method should be required in the aquatic fields. To address this, we developed a field-ready cyanobacterial pretreatment device and an electrochemical clustered regularly interspaced short palindromic repeats (EC-CRISPR) biosensor. The cyanobacterial pretreatment device consists of a syringe, glass bead, and graphene oxide (GO) bead. Then, the M. aeruginosa dissolved in the freshwater sample was added to fabricated filter. After filtration, the purified gene was loaded onto a CRISPR-based electrochemical biosensor chip to detect M. aeruginosa gene fragments. The biosensor was composed of CRISPR/Cpf1 protein conjugated with MXene on an Au microgap electrode (AuMGE) integrated into a printed circuit board (PCB). This AuMGE/PCB system maximizes the signal-to-noise ratio, which controls the working and counter electrode areas requiring only 3 µL samples to obtain high reliability. Using the extracted M. aeruginosa gene with a pre-treatment filter, the CRISPR biosensor showed a limit of detection of 0.089 pg/µl in fresh water. Moreover, selectivity test and matrix condition test carried out using the EC-CRISPR biosensor. These handheld pre-treatment kit and biosensors can enable field-ready detection of CyanoHABs.

RGO-PANI composite Au microelectrodes for sensitive ECIS analysis of human gastric (MKN-1) cancer cells.

Microelectrode-based cell chip studies for cellular responses often require improved adhesion and growth conditions for efficient cellular diagnosis and high throughput screening in drug discovery. Cell-chip studies are often performed on gold electrodes due to their biocompatibility, and stability, but the electrode-electrolyte interfacial capacitance is the main drawback to the overall sensitivity of the detection system. Thus, here, we developed reduced graphene oxide-polyaniline-modified gold microelectrodes for real-time impedance-based monitoring of human gastric adenocarcinoma cancer (MKN-1) cells. The impedance characterization on modified electrodes showed 28-fold enhanced conductivity than the bare electrodes, and the spectra were modeled with proper equivalent circuits to extrapolate the values of circuit elements. The impedance of both time-and frequency-dependent measurements of cell-covered modified electrodes with equivalent model circuits was analyzed to achieve cellular behavior, such as adhesion, spreading, proliferation, and influence of anti-cancer agents. The normalized impedance at 41.5 kHz (|Z|norm 41 kHz) was selected to monitor the cell growth analysis, which was found linear with the proliferation of adherent cells along with the influence of the anticancer drug agent on the MKN-1 cells. The synergistic effects and biocompatible nature of PANI-RGO modifications improved the overall sensitivity for the cell-growth studies of MKN-1 cells.

Impact of intensive rehabilitation on long-term prognosis after stroke: A Korean nationwide retrospective cohort study.

An increasing number of patients are receiving rehabilitation after stroke. But the impact of intensive rehabilitation on the long-term prognosis of patients with stroke remains to be elucidated. The purpose of this study was to identify the impact of intensive rehabilitation on the long-term prognosis of patients with stroke using data from the National Health Insurance Service database. This is a register-based, retrospective cohort study. Using data from the National Health Insurance Service database, we included the patients who received rehabilitation for stroke from 2006 to 2013. Of the 14,984 patients diagnosed with stroke, 2483 died within 1 year, and 2866 did not receive rehabilitation; hence, they were also excluded. The final sample included 9635 (49.2% men, 50.8% women) patients. After correcting for covariates, the Cox model was used to evaluate the effects of physical therapy (PT) and occupational therapy (OT) on survival. We estimated the independent contribution of each factor to the risk of death from the initiation of rehabilitation. Significant differences in mortality were observed according to age, Charlson comorbidity index (CCI), income level, and stroke type. Patients with stroke who received both PT and OT had a better long-term prognosis than those who received either treatment alone. Therapy performed by a physical therapist with more than 120 hours of training effectively improved the patients' long-term prognosis. Intensive PT and OT will help improve the long-term prognosis of patients with stroke. This study emphasizes the importance of intensive rehabilitation in these patients.

Differences in the gut microbiome composition of Korean children and adult samples based on different DNA isolation kits.

Recent studies have revealed that the composition of human gut microbiota varies according to region, race, age, diet, living environment, and sampling and DNA extraction method. The purpose of this study was to broaden our understanding of the intestinal microbial composition of Koreans by conducting a 16S rRNA amplicon sequencing on 78 Korean samples composed of adults, children, normal and obese groups. We compared the microbiome composition and diversity of these groups at different levels including the phylum and genus level using two different stool DNA extraction kits of QIAamp® PowerFecal® DNA Kit (Qiagen, Hilden, Germany) and CT Max Fecal DNA Kit (Ct bio, Korea). We found that Ct bio (Ct) kit recovered higher DNA yields and OTUs than QIAamp® PowerFecal® DNA Kit (Qia). The Ct kit, which adopted more rigorous bead beating method, detected the most Gram-positive (G+) bacteria, Firmicutes, at the Phylum level, whereas the Qia kit, which used a less rigorous cell lysis method, found the most Gram-negative (G-) bacteria, Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio showed no significant difference between the obese and the normal groups of same kit; however, they were significantly different with two different kits. There was a difference in the intestinal flora between healthy Korean adults and children. The taxa that differed significantly between the adults and children were Bacteroides, Bifidobacterium, Prevotella, and Subdoligranulum. There was no significant difference in the intestinal flora between the normal weight group and the obese group in adults and children, respectively. This is probably because the difference in body mass index (BMI) between the sample groups collected in this study is statistically significant, but it is not large enough to show a clear difference in the flora. Therefore, these results should be interpreted with caution while considering the BMI values and Korean obesity criterion together.

Facile and foldable point-of-care biochip for nucleic acid based-colorimetric detection of murine norovirus in fecal samples using G-quadruplex and graphene oxide coated microbeads.

Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.

Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification.

It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.

Mismatch-introduced DNA probes constructed on the basis of thermodynamic analysis enable the discrimination of single nucleotide variants.

Genotyping of single nucleotide variants (SNVs) has enabled the assessment of disease-related risk factors and significantly improved the potency of diagnosis and prognosis. Meanwhile, genotyping of SNVs is challenging due to the high sequence similarity between wild-type (WT) and SNV. To increase the discrimination between WT and SNV, probes are modified with nucleic acid analogues such as locked nucleic acid (LNA), or deliberate mismatches are introduced to the probe sequence. However, nucleic acid analogues have limitation in high cost and complexity in their synthesis. And a generalized methodology has not been proposed for determining the position and type of deliberate mismatches at the designated experimental conditions to the best of our knowledge. Herein, we propose a reliable workflow for designing mismatch-introduced probes (MIPs) based on nucleic acid thermodynamic analysis and rejection sampling. The theoretical hybridization state of MIP was calculated using nucleic acid thermodynamics, and the detectability was estimated by rejection sampling that simulates the errors from experimental environments. We fabricated MIPs for SNVs in epidermal growth factor receptor, and experimentally demonstrated optimized detectability. The detectability increased up to 7.19-fold depending on the position and type of mismatch; moreover, the optimized MIP showed higher detectability than the LNA probe. This indicates that the workflow can be broadly applied to the optimization of probe sequence for the detection of various disease-related SNVs.

Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/µL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

Discrimination and isolation of the virus from free RNA fragments for the highly sensitive measurement of SARS-CoV-2 abundance on surfaces using a graphene oxide nano surface.

It is highly important to sensitively measure the abundance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on various surfaces. Here, we present a nucleic acid-based detection method consisting of a new sample preparation protocol that isolates only viruses, not the free RNA fragments already present on the surfaces of indoor human-inhabited environments, using a graphene oxide-coated microbead filter. Wet wipes (100 cm2), not cotton swabs, were used to collect viruses from environmental surfaces with large areas, and viruses were concentrated and separated with a graphene oxide-coated microbead filter. Viral RNA from virus was recovered 88.10 ± 8.03% from the surface and free RNA fragment was removed by 99.75 ± 0.19% from the final eluted solution. When we tested the developed method under laboratory conditions, a 10-fold higher viral detection sensitivity (Detection limit: 1 pfu/100 cm2) than the current commercial protocol was observed. Using our new sample preparation protocol, we also confirmed that the virus was effectively removed from surfaces after chemical disinfection; we were unable to measure the disinfection efficiency using the current commercial protocol because it cannot distinguish between viral RNA and free RNA fragments. Finally, we investigated the presence of SARS-CoV-2 and bacteria in 12 individual negative pressure wards in which patients with SARS-CoV-2 infection had been hospitalized. Bacteria (based on 16 S DNA) were found in all samples collected from patient rooms; however, SARS-CoV-2 was mainly detected in rooms shared by two patients.

Electrospun Nanofibers Embedded with Copper Oxide Nanoparticles to Improve Antiviral Function.

Many studies on anti-bacterial/antiviral surfaces have been conducted to prevent epidemic spread worldwide. Several nanoparticles such as those composed of silver and copper are known to have antiviral properties. In this study, we developed copper oxide (CuO) nanoparticle-incorporated nanofibers to inactivate or remove viruses. The CuO nanoparticle-incorporated nanofiber was fabricated with a hydrophobic polymer-polyvinylpyrrolidone (PVP)-using electrospinning, and CuO nanoparticles were exposed from the PVP polymer surface by etching the nanofiber with oxygen plasma. The fabrication conditions of electrospinning and oxygen plasma etching were investigated by scanning electron microscopy (SEM), and field emission transmission electron microscopy (FETEM)/ energy dispersive spectrometry (EDS). H1N1 virus was utilized as the target sample and quantified by RT-qPCR. The antiviral efficacy of CuO nanoparticle-incorporated nanofibers was compared against bare CuO nanoparticles. Overall, 70% of the viruses were inactivated after CuO nanoparticle-incorporated nanofibers were incubated with 10² pfu/mL of H1N1 virus solution for 4 h. This indicates that the developed CuO nanoparticle-incorporated nanofibers have noticeable antiviral efficacy. As the developed CuO nanoparticle-incorporated nanofibers exerted promising antiviral effects against H1N1 virus, it is expected to benefit global health by preventing epidemic spread.

Bacterial Isolation by Adsorption on Graphene Oxide from Large Volume Sample.

Graphene oxide (GO) is a well-known two-dimensional nanomaterial with broad applications in various fields. In particular, the functional groups of GO has demonstrated significance in the molecular binding interactions. GO is normally coated on a solid surface as it is difficult to handle due to its nano-scaled size. Therefore, chemical properties of surface-coated GO depend on the morphological structure of GO on the surface and the operating conditions during the coating process. Isolation of bacteria from environmental samples such as river and pond water is important for increasing the analytical sensitivity of sensor devices. The main issue in isolation of bacteria from an environmental sample is adsorption capacity per unit time. However, increasing the velocity of water sample to elevate the process rate induces high shear stress on the surface, such that the bacteria adsorption rate on the surface is reduced. In this study, we investigated the morphological and chemical properties of sonicated GO and GO-coated surface by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The sonicated GO-coated beads were successfully used for concentrating bacteria from a large-volume sample as opposed to the conventional methods. It can be concluded that, GO-coated surfaces are prospective platforms for concentrating bacteria from various samples and play a major role in reducing the concentration time.

Improvement of Heat Sink Effect Using Zinc Oxide Nanostructure.

Heat sinks that dissipate heat effectively play a significant role in devices with high-precision temperature control, such as thermal cyclers for polymerase chain reaction (PCR). This study was carried out to develop a heat sink with a high thermal conductivity to dissipate heat effectively. To increase the surface area of the heat sink, zinc oxide (ZnO) nanostructures were fabricated on an aluminum plate. ZnO nanostructures were fabricated by hydrothermal method and confirmed by scanning electron microscopy and X-ray diffraction. With the increase in the concentration of the precursors, the length of the nanorods increased, and with longer reaction time, nanostructures connected with higher stability and larger surface area. Thermal conductivity is increased by ZnO nanostructures and is affected by the concentration of precursors and the reaction time. Thermal conductivity of an optimal ZnO-coated Al plate is 2 times higher than that of a bare one. This technology can be applied to portable PCR devices to reduce weight, size, and power consumption.

Integrated microsystems for the in situ genetic detection of dengue virus in whole blood using direct sample preparation and isothermal amplification.

Owing to the frequent outbreak of dengue fever worldwide, a highly sensitive but in situ simple process diagnostic device is required to detect the dengue virus. However, the current immune affinity-based methods have sensitivity issues and nucleic acid-based diagnostic devices have not been suitable for field diagnosis due to the complexity in sample preparation. Here, a simple and fast nucleic acid-based diagnostic tool to directly detect dengue viruses in whole blood is demonstrated using a microbead-assisted direct sample preparation buffer (MB-buffer) and isothermal amplification (loop-mediated isothermal amplification, LAMP). To maximize the performance of the sample preparation process in the microfluidic chip platform, the chemical composition of the sample preparation buffer is simplified and combined with physical tools (heating and bead beating). The entire serial processes consisted of only (1) sample (whole blood) loading, (2) stirring for 90 s, (3) heating at 70 °C for 10 min, and (4) LAMP amplification in the simply designed microfluidic chip cartridge. A single syringe was utilized for sample loading and microfluidic solution transfer. Consequently, dengue viruses were qualitatively detected and discriminated with high sensitivity (LOD: 102 PFU per 200 µL of whole blood) in less than 1 hour without the use of any sophisticated system.

Semi-automatic instrumentation for nucleic acid extraction and purification to quantify pathogens on surfaces.

Public lavatories may cause the spread of infectious pathogens because they are enclosed spaces that both healthy people and patients can use. Thus, surface analysis for microbial contamination in public lavatories is of great importance because it is considered as an indicator of hygiene control. Herein, we developed polymerase chain reaction (PCR)-compatible surface sample preparation tools to increase the detection sensitivity and reproducibility within a short time using a semi-automatic detection system. The bacteria and viruses on different surfaces were collected using half A4-sized wipes. The wipes were treated through four different processes in a cartridge: (1) the pathogens were transferred from the wipes to the aqua phase using simple gentle vortexing; (2) the bacteria and viruses were concentrated by adsorption on the graphene surface; (3) the pathogens on the graphene layer were perfectly lysed using bead-beating tools and (4) the released DNA/RNA was collected in a microtube. The prepared nucleic acid sample was amplified using PCR or loop-mediated isothermal amplification (LAMP). At least one order of magnitude higher sensitivity was achieved using the wipe collecting method compared to that achieved using the normal swab method. This was confirmed using a semi-automatic cartridge for the wipe sampling in a lavatory hygiene test.

Nanostructured Au-Pt hybrid disk electrodes for enhanced parathyroid hormone detection in human serum.

Most clinical tests for biomarker detection require the support of a laboratory, and the results are usually slow, less sensitive, and lack the possibility for Point-of-Care (PoC) testing. Further, with the increasing demand for sensitive, portable, rapid, and low-cost devices for clinical PoC applications, innovative methods are crucial. Thus, we report on utilizing nanostructured gold-platinum (Au-Pt) hybrid electrodes as a PoC device for highly sensitive and selective PTH detection in human serum samples. The method employs the immobilization of 3-mercaptopropionic acid to Au and subsequent activation of the carboxyl groups to enable anti-PTH antibody immobilization. Serum PTH was detected by monitoring the changes in electrochemical properties (ΔRct and Δi) of the sensor using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) against a standard hexacyanoferrate (II/III) probe. Changes in relative response percentage (RR%) in electrochemical properties due to increased PTH concentrations in serum were observed with EIS and DPV. The biosensor exhibited a low detection limit of 0.36 pg.mL-1 (EIS) and 0.59 pg.mL-1 (DPV) in serum with a linear range of 1 to 100,000 pg.mL-1. Further, to validate the accuracy of the proposed method, clinical samples (n = 20) were examined using the EIS method and compared to an established commercial test.

Label-Free Impedance Sensing of Aflatoxin B1 with Polyaniline Nanofibers/Au Nanoparticle Electrode Array.

Aflatoxin B1 (AFB1) is produced by the Aspergillus flavus and Aspergillus parasiticus group of fungi which is most hepatotoxic and hepatocarcinogenic and occurs as a contaminant in a variety of foods. AFB1 is mutagenic, teratogenic, and causes immunosuppression in animals and is mostly found in peanuts, corn, and food grains. Therefore, novel methodologies of sensitive and expedient strategy are often required to detect mycotoxins at the lowest level. Herein, we report an electrochemical impedance sensor that selectively detects AFB1 at the lowest level by utilizing polyaniline nanofibers (PANI) coated with gold (Au) nanoparticles composite based indium tin oxide (ITO) disk electrodes. The Au-PANI nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) spectroscopy, and electrochemical impedance spectroscopy (EIS). The composite electrode exhibited a 14-fold decrement in |Z|1Hz in comparison with the bare electrode. The Au-PANI acted as an effective sensing platform having high surface area, electrochemical conductivity, and biocompatibility which enabled greater loading deposits of capture antibodies. As a result, the presence of AFB1 was screened with high sensitivity and stability by monitoring the changes in impedance magnitude (|Z|) in the presence of a standard iron probe which was target specific and proportional to logarithmic AFB1 concentrations (CAFB1). The sensor exhibits a linear range 0.1 to 100 ng/mL with a detection limit (3) of 0.05 ng/mL and possesses good reproducibility and high selectivity against another fungal mycotoxin, Ochratoxin A (OTA). With regard to the practicability, the proposed sensor was successfully applied to spiked corn samples and proved excellent potential for AFB1 detection and development of point-of-care (POC) disease sensing applications.

Elimination of Humic Acid from Aqueous Sample Using Zinc Oxide/Graphene Oxide-Coated Microbeads.

Natural organic matter (NOM) is known to cause major problems with drinking water quality management, such as sedimentation of disinfectants during the purification process, microbial growth of water pipes, and corrosion of pipes. For efficient and continuous removal of NOM from drinking water, a packed bed-type platform containing microbeads based on nanostructured zinc oxide (ZnO) was developed. ZnO was synthesized on graphene oxide (GO)-coated microbeads by optimizing the ZnO concentration and reaction time. The morphology of the synthesized ZnO-coated microbeads was confirmed by scanning electron microscopy, and an adsorption test was conducted using a cationic dye. The ZnO/GO microbeads were packed in a microtube. A humic acid contaminated aqueous solution was allowed to flow through the microbeads, and its removal rate was measured by UV-vis spectroscopy. This study confirmed that the purification platform containing ZnO removed more than 90% of humic acid of about 1,000 ppm.

Short-Length DNA Adsorption on Graphene Oxide-Coated Microbeads for DNA Target Separation from Clinical Samples.

Nucleic acid preparation (concentration and purification of various nucleic acid targets) from biological samples is essential for personalized and precision medicine. The adsorption of short-length DNA on graphene oxide (GO) layers was investigated and compared with that on silica surfaces. GO was efficiently coated on glass beads to be used more easily and spatially. Surface of the GO bead was confirmed by field-emission scanning electron microscopy. GO-coated beads were packed and the adsorption conditions of short-length DNA were optimized under various pH and flow rate conditions. The amount of adsorbed DNA was confirmed by real-time polymerase chain reaction and visualized using fluorescence microscopy.

Fabrication of peptide stabilized fluorescent gold nanocluster/graphene oxide nanocomplex and its application in turn-on detection of metalloproteinase-9.

This study introduces the double-ligands stabilizing gold nanoclusters and the fabrication of gold nanocluster/graphene nanocomplex as a "turn-on" fluorescent probe for the detection of cancer-related enzyme matrix metalloproteinase-9. A facile, one-step approach was developed for the synthesis of fluorescent gold nanoclusters using peptides and mercaptoundecanoic acid as co-templating ligands. The peptide was designed to possess a metalloproteinase-9 cleavage site and to act not only as a stabilizer but also as a targeting ligand for the enzyme detection. The prepared gold nanoclusters show an intense red fluorescence with a broad adsorption spectrum. In the presence of the enzyme, due to the excellent quenching properties and the negligible background of graphene oxide, the developed peptide-gold nanocluster/graphene nanocomplex yielded an intense "turn-on" fluorescent response, which strongly correlated with the enzyme concentration. The limit of detection of the nanocomplex was 0.15nM. The sensor was successfully applied for "turn-on" detection of metalloproteinase-9 secreted from human breast adenocarcinoma MCF-7 cells with high sensitivity, selectivity, significant improvement in terms of detection time and simplicity.