Bioengineering Laboratory

We report that gold/zinc oxide (Au/ZnO) nanocomposite films were effectively employed to enhance the performance of surface plasmon resonance (SPR) for the detection of tumor markers. Carbohydrate antigen 15.3 (CA15-3), a tumor marker for breast cancer, was chosen as a model analyte. We analyzed intensity response to the samples at various concentrations (0.0125 U/mL to 160 U/mL) in pleural fluid to evaluate the detection capability of the SPR biosensor based on Au/ZnO thin films. The linear range extended from 1 to 40 U/mL with a correlation coefficient of R 2 ) 0.991 and a limit of detection reaching 0.025 U/mL at a signal-to-noise ratio of 3:1. Compared with the degree of the shift in SPR intensity induced by the specific binding event between antibody and antigen, the change of intensity on the Au/ZnO layers was increased by at least 2 fold over that on the gold/chromium (Au/Cr) layers. In addition, we determined that the Au/ZnO layers allowed for a detection limit 4 times lower than the Au/Cr layers, which are in widespread use as the sensing interfaces in current SPR-based detectors. In conclusion, the use of Au/ ZnO films greatly enhanced the SPR signal yield for this bimolecular interaction and showed high sensitivity.

In this paper, an improvement method for detection of tumor marker with a label-free biosensor is reported. The polyethylene glycol (PEG)-modified chitosan hydrogel as a biosensing interfacial architecture was formed on the gold surface and its potential was evaluated with a quartz crystal microbalance (QCM). This hydrogel was modified to increase its negative charges and then use electrostatic interactions with the positive charges of the amine surfaces. The biosensing application was investigated by use of the antibody-antigen model system of carbohydrate antigen 15-3 (CA15-3), an important indicator in the prognosis of breast cancer. Compared to the covalent immobilization method, the QCM biosensor with the proposed immobilization showed improved sensitivity. In addition, the regeneration of the developed immunosensor was obtained after the release by rinsing the sensor with glycine/HCl solution. Based on these results, it was believed that such a three-dimensional polyelectrolyte multilayer can be applied to the development of immunoassays.

Preterm birth is the main cause of perinatal morbidity and mortality throughout the world. Fetal fibronectin (fFN) is one type of glycoprotein detected in the interface of the choriodecidual junction. Preterm labor is related to the disruption of the choriodecidual junction and thus fFN releases in the ectocervix or posterior vaginal fornix. Quantization of fFN can assist the diagnosis and prevention of preterm birth.

Inorganic mercury ion (Hg 2+ ) has been shown to coordinate to DNA duplexes that feature thymine-thymine (T-T) base pair mismatches. This observation suggests that an Hg 2+ -induced conformational change in a single-stranded DNA molecule can be used to detect aqueous Hg 2+ . Here, we have developed an analytical method using surface plasmon resonance (SPR) to develop a highly selective and sensitive detection technique for Hg 2+ that takes advantage of T-Hg 2+ -T coordination chemistry. The general concept used in this approach is that the "turn-on" reaction of a hairpin probe via coordination of Hg 2+ by the T-T base pair results in a substantial increase in the SPR response, followed by specific hybridization with a gold nanoparticle probe to amplify the sensor performance. Meanwhile, the limit of detection is 1 nM, which is lower than other recently developed techniques. A linear correlation is observed between the measured SPR reflectivity and the logarithm of the Hg 2+ concentration over the concentration range of 5-5000 nM. Additionally, the SPR system provides high selectivity for Hg 2+ in the presence of other divalent metal ions up to micromolar concentration levels. The proposed approach is also successfully utilized for the determination of Hg 2+ in water samples.

We report the development of a surface plasmon resonance sensor based on the silver ion (Ag+)-induced conformational change of a cytosine-rich, single-stranded DNA for the detection of Ag+ and cysteine (Cys) in aqueous solutions. In the free state, single-stranded oligonucleotides fold into double-helical structures through the addition of Ag+ to cytosine–cytosine (C–C) mismatches. However, in the presence of Cys, which competitively binds to Ag+, the formation of the C–Ag+–C assembly is inhibited, resulting in free-state, single-stranded oligonucleotides. To enhance sensitivity, the DNA intercalator, daunorubicin, was employed to achieve signal enhancement. The detection limit for Ag+ was 10 nM with a measurement range of 50–2,000 nM, and the detection limit for Cys was 50 nM with a measurement range of 50–2,000 nM. This simple assay was also used to individually determine the spiked Ag+ concentration in water samples and Cys concentrations in biological fluid samples.

Tubercle bacillus [TB] is one of the most important chronic infectious diseases that cause millions of deaths annually. While conventional smear microscopy and culture methods are widely used for diagnosis of TB, the former is insensitive, and the latter takes up to 6 to 8 weeks to provide a result, limiting the value of these methods in aiding diagnosis and intermediate decisions on treatment. Therefore, a rapid detection method is essential for the diagnosis, prognosis assessment, and recurrence monitoring. A new surface plasmon resonance [SPR] biosensor based on an array format, which allowed immobilizing nine TB antigens onto the sensor chip, was constructed. Simultaneous determination of multiple TB antibodies in serum had been accomplished with this array-based SPR system. The results were compared with enzyme-linked immunosorbent assay, a conventional immunological method. Array-based SPR showed more advantages in providing label-free and real-time detection. Additionally, the high sensitivity and specificity for the detection of TB infection showed its potential for future development of biosensor arrays for TB diagnosis.

Preterm birth is the leading cause of perinatal morbidity and mortality. Fetal fibronectin (fFN), a glycoprotein in the extracellular matrix of the amniotic membranes, is the most powerful biomarker for predicting the risk of preterm birth. Biosensors using the surface plasmon resonance (SPR) response are potentially useful in quantitatively measuring molecules. We established a standard calibration curve of SPR intensity against fFN concentration and used the SPR-based biosensor to detect fFN concentrations in the cervicovaginal secretions of pregnant women between 22 and 34 weeks of gestation. The calibration curve extends from 0.5 ng/mL to 100 ng/mL with an excellent correlation (R 2 = 0.985) based on standard fFN samples. A cutoff value of 50 ng/mL fFN concentration in commercial ELISA kits corresponds to a relative intensity of 17 arbitrary units (a.u.) in SPR. Thirty-two pregnant women were analyzed in our study. In 11 women, the SPR relative intensity was greater than or equal to 17 a.u., and in 21 women, the SPR relative intensity was less than 17 a.u. There were significant differences between the two groups in regular uterine contractions (p = 0.040), hospitalization for tocolysis (p = 0.049), and delivery weeks (p = 0.043). Our prospective study concluded that SPR-based biosensors can quantitatively measure fFN concentrations. These results reveal the potential utility of SPR-based biosensors in predicting the risk of preterm birth.

Interferon-gamma (IFN-g) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-g, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-g with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-g binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-g induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.

Heavy metals greatly in°uence animal physiology, even at small doses. Among these metals, the copper ion is of great concern due to its e®ects on humans and wide applications in industry. Compared to atomic absorption spectroscopy and inductively coupled plasma-mass spectrometry, which destroy the samples that are analyzed, optical techniques do not decompose the analyte and have become a popular¯eld of recent research. In this paper, we combined a novel optical detector that did not require sample-labeling, called surface plasmon resonance (SPR), with chitosan to detect copper ions by modifying the functional groups of chitosan through pH modi¯cation. Compared to other optical detectors, the SPR system was relatively fast and involved fewer experimental confounding factors. The three-dimensional structure of chitosan was used to obtain lower detection limits. Moreover, modi¯cation of the chitosan functional groups resulted in e±cient regeneration by controlling the pH. A detection limit of 0.1 M was obtained (linear range: 0.5À10 M, R 2 ¼ 0:976), and the speci¯city was certi¯ed by comparing the copper ion with six other ions. Additionally, we successfully regenerated the SPR chips by modifying the functional groups. In conclusion, the chitosanÀSPR system detected copper ions with improved detection limits using a quick and simple regeneration method.

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody-protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.

ELISA and ELISPOT methods are utilized for interferon-gamma (IFN-γ) release assays (IGRAs) to detect the IFN-γ secreted by T lymphocytes. However, the multi-step protocols of the assays are still performed with laboratory instruments and operated by well-trained people. Here, we report a membrane-based microfluidic device integrated with a surface plasmon resonance (SPR) sensor to realize an easy-to-use and cost effective multi-step quantitative analysis. To conduct the SPR measurements, we utilized a membrane-based SPR sensing device in which a rayon membrane was located 300 μm under the absorbent pad. The basic equation covering this type of transport is based on Darcy's law. Furthermore, the concentration of streptavidin delivered from a sucrose-treated glass pad placed alongside the rayon membrane was controlled in a narrow range (0.81 μM ± 6%). Finally, the unbound molecules were removed by a washing buffer that was pre-packed in the reservoir of the chip. Using a bi-functional, hairpin-shaped aptamer as the sensing probe, we specifically detected the IFN-γ and amplified the signal by binding the streptavidin. A high correlation coefficient (R 2 = 0.995) was obtained, in the range from 0.01 to 100 nM. A detection limit of 10 pM was achieved within 30 min. Thus, the SPR assay protocols for IFN-γ detection could be performed using this simple device without an additional pumping system.