Hyoyoung Mun

An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1.

A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn.

A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A.

Binding of an analyte on the surface of a nanoparticle typically promotes a change in the local refractive index, which gives rise to a shift in the wavelength of the localized surface plasmon resonance (LSPR) absorption band. The magnitude of the LSPR wavelength change is dependent on both the location of the analyte relative to the surface of the nanoparticle and the degree of alteration of the refractive index. We have employed this phenomenon as the basis for designing a new, label-free approach for the detection of the toxic mold mycotoxin, ochratoxin A (OTA) that employs a gold nanorod (GNR) and an aptamer target binding mechanism. In this system, binding of OTA causes an accumulation of OTA and G-quadruplex structure of the aptamer. This process results in a longitudinal wavelength shift of the LSPR peak associated with a change in the local refractive index near the GNR surface. By using this method, OTA can be quantitatively detected at concentrations lower than 1 nM. In addition, the results of this effort show that aptamer functionalized GNR substrate is robust in that it can be regenerated for reuse over seven times by heating in methanol at 70 °C to remove OTA. Moreover, the proposed biosensor system exhibits high selectivity for OTA over other mycotoxins. Finally, the sensor can be employed to detect OTA in ground corn samples with excellent recovery levels.

Homogeneous Immunosensor Based on Luminescence Resonance Energy Transfer for Glycated Hemoglobin Detection Using Upconversion Nanoparticles

We report an immunosensor based on luminescence resonance energy transfer (LRET) to detect homogeneous glycated hemoglobin (HbA1c). This system uses near-infrared (NIR)-to-visible rare-earth upconversion nanoparticles (UCNPs), such as NaYF4:Yb(3+), Er(3+), as the donor and HbA1c as the acceptor. The HbA1c used as target molecules showed absorption at 541 nm, which corresponded with the emission of the UCNPs. When HbA1c was added, LRET occurred between the donor and acceptor under laser irradiation of 980 nm because of the specific recognition between the anti-HbA1c monocolonal antibody-functionalized UCNPs and HbA1c. In the absence of HbA1c, there was strong upconversion luminescence intensity; however, in its presence, the distance between the donor and acceptor decreased to enable energy transfer, consequently quenching the luminescence of the UCNPs. The proposed method was successfully applied to HbA1c detection in blood samples. Our results indicate that the LRET-based immunosensor allows for specific and sensitive detection of HbA1c in a homogeneous manner.

Colorimetric detection of PCR products of DNA from pathogenic bacterial targets based on a simultaneously amplified DNAzyme

AbstractA novel strategy was devised for colorimetric analysis of the products of the polymerase chain reaction (PCR). The method takes advantage of simultaneous amplification of a horseradish peroxidase-mimicking DNAzyme (HRPzyme) during the PCR process. It is performed using a DNA specific forward primer and a universal reverse primer containing a complementary HRPzyme sequence. The double-strand PCR products, which include the HRPzyme sequence, are treated with a mixture of hemin and TMB (3,3′,5,5′–tetramethylbenzidine) in the presence of hydrogen peroxide. The resulting HRPzyme/hemin complex then promotes a peroxidase mimicking reaction, which produces the blue colored oxidized TMB. This colorimetric method can be more easily performed than previously developed gel based detection procedures and, as a result, can be conveniently applied to the specific and sensitive colorimetric analysis of DNA sequences arising from pathogenic bacteria. The potentially broad applicability of the new method has been demonstrated by its use in the identification of the 16s rDNA of Salmonella Typhimurium. FigureA novel strategy was devised for simple colorimetric analysis of PCR products with amplification of a horseradish peroxidase-mimicking DNAzyme(HRPzyme). This colorimetric method can be much more easily performed than previously developed gel based detection procedures and potentially broad applicability for other DNA analysis.

Colorimetric Detection of Norovirus in Oyster Samples through DNAzyme as a Signaling Probe

Worldwide, norovirus is one of the most associated causes of acute gastroenteritis, which leads to nearly 50 000 child deaths every year in developing countries. Therefore, there is great demand to develop a rapid, low-cost, and accurate detection assay for the foodborne norovirus infection to reduce mortality caused by norovirus. Considering the importance of norovirus, we have demonstrated a highly sensitive and specific colorimetric detection method for analysis of human norovirus genogroups I and II (HuNoV GI and GII) in oyster samples. This is the first report to employ colorimetric HRPzyme-integrated polymerase chain reaction (PCR) for direct norovirus detection from the real shellfish samples. We found that the HRPzyme-integrated PCR method is more sensitive than the gel electrophoresis approach and could detect the HuNoV GI and GII genome up to 1 copy/mL. The specificity of the proposed method was successfully demonstrated for HuNoV GI and GII. Further, we performed testing HuNoVs in the spiked oyster samples, and the HRPzyme-integrated PCR method proved to be an ultrasensitive and selective method for detecting HuNoVs in the real samples. By integration of the proposed method with the portable PCR machine, it would be more reliable to improve food safety by detecting HuNoVs in the different types of shellfish, such as oyster and mussel, at the production field.

Ultrasensitive Detection of Escherichia coli O157:H7 by Immunomagnetic Separation and Selective Filtration with Nitroblue Tetrazolium/5-Bromo-4-chloro-3-indolyl Phosphate Signal Amplification

Here, we report an enhanced colorimetric method using enzymatic amplification with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation for the ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective filtration. Biotinylated anti- E. coli O157:H7 antibody and streptavidin-alkaline phosphatase were conjugated to the surface of magnetic nanoparticles, and E. coli O157:H7-conjugates complexes remained on the membrane filter surface. The resultant light brown spots on the membrane filter were amplified with NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with an increasing E. coli O157:H7 concentration. E. coli O157:H7 was detected in pure samples with limits of detection of 10 and 6.998 colony-forming units (CFU)/mL through visual observation and measurement of optical density, respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli O157:H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This enhanced colorimetric method has potential for on-site detection of food contaminants and environmental pollutants.

Single-Step LRET Aptasensor for Rapid Mycotoxin Detection

Contamination of foods by mycotoxins is a common yet serious problem. Owing to the increase in consumption of fresh produce, consumers have become aware of food safety issues caused by mycotoxins. Therefore, rapid and sensitive mycotoxin detection is in great demand in fields such as food safety and public health. Here we report a single-step luminescence resonance energy transfer (LRET) aptasensor for mycotoxin detection. To accomplish the single-step sensor, our sensor was constructed by linking a quencher-labeled aptamer through a linker to the surface of upconversion nanoparticles (UCNPs). Our LRET aptasensor is composed of Mn2+-doped NaYF4:Yb3+,Er3+ UCNPs as the LRET donor, and black hole quencher 3 (BHQ3) as the acceptor. The maximum quenching efficiency is obtained by modulating the linker length, which controls the distance between the quencher and the UCNPs. Our distinctive design of LRET aptasensor allows detection of mycotoxins selectively in colored food samples within 10 min without multiple bioassay steps. We believe our single-step aptasensor has a significant potential for on-site detection of food contaminants, environmental pollutants, and biological metabolites.

Adenosine Triphosphate Bioluminescence-Based Bacteria Detection Using Targeted Photothermal Lysis by Gold Nanorods

Bacterial infections are common causes of morbidity and mortality worldwide; therefore, environmental contamination by bacterial pathogens represents a global public health concern. Consequently, a selective, rapid, sensitive, and in-field detection platform for detecting significant bacterial contamination is required to ensure hygiene and protect public health. Here, we developed a fast and simple platform for the selective and sensitive detection of bacteria by measuring adenosine triphosphate (ATP) bioluminescence following targeted photothermal lysis mediated by antibody-conjugated gold nanorods. This method employed both targeted photothermal lysis of bacteria by near-infrared (NIR) irradiation and highly selective detection of the lysed bacteria via ATP bioluminescence within 36 min (incubation, 30 min; NIR irradiation, 6 min). The use of the proposed method allowed limits of detection in pure solution of 12.7, 70.7, and 5.9 CFU for Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes, respectively. Additionally, bacteria were successfully detected on artificially inoculated plastic cutting boards. Furthermore, this method was highly specific, without cross-reaction among pathogenic bacteria. We believe that the proposed method has significant potential as an on-site diagnostic tool for applications associated with public health and environmental pollution monitoring.

Light shift from ultraviolet to near infrared light: Cerenkov luminescence with gold nanocluster - near infrared (AuNc-NIR) conjugates

Cerenkov luminescence (CL) is generated when a charged particle moves faster than the speed of light in dielectric media. Recently CL imaging becomes an emerging technique with the use of radioisotopes. However, due to relatively weak blue light production and massive tissue attenuation, CL has not been applied widely. Therefore, we attempted to shift the CL emission to more near infrared (NIR) spectrum for better tissue penetration by using Cerenkov Radiation Energy Transfer (CRET). Gold nanoclusters were conjugated with NIR dye molecules (AuNc-IR820 and AuNc-ICG) to be activated with ultraviolet light. We found optimal conjugate concentrations of AuNc-NIR conjugates by spectroscopy system to generate maximal photon emission. When exposed by ultraviolet light, the emission of NIR light from the conjugates were verified. In quantitative analysis, AuNc-NIR conjugates emit brighter light signal than pure AuNc. This result implies that NIR fluorescent dyes (both IR820 and ICG) can be excited by the emission from AuNc. Following the above baseline experiment, we mixed F-18 fluorodeoxyglucose (F-18 FDG) radioisotope to the AuNc- NIR conjugates, to confirm NIR emission induced from Cerenkov radiation. Long pass filter was used to block Cerenkov luminescence and to collect the emission from AuNc-NIR conjugates. Instead of one long exposure imaging with CCD, we used multiple frame scheme to eliminate gamma radiation strike in each frame prior to combination. In summary, we obtained NIR emission light from AuNc-NIR conjugated dyes that is induced from CL. We plan to perform in vivo small animal imaging with these conjugates to assess better tissue penetration.