Ying-Feng Chang

Diagnostic detection of human lung cancer-associated antigen using a gold nanoparticle-based electrochemical immunosensor.

The development of rapid and sensitive methods for the detection of immunogenic tumor-associated antigen is important not only for understanding their roles in cancer immunology but also for the development of clinical diagnostics. Alpha-enolase (ENO1), a p48 molecule, is widely distributed in a variety of tissues, whereas gamma-enolase (ENO2) and beta-enolase (ENO3) are found exclusively in neuron/neuroendocrine and muscle tissues, respectively. Because ENO1 has been correlated with small cell lung cancer, nonsmall cell lung cancer, and head and neck cancer, it can be used as a potential diagnostic marker for lung cancer. In this study, we developed a simple, yet novel and sensitive, electrochemical sandwich immunosensor for the detection of ENO1; it operates through physisorption of anti-ENO1 monoclonal antibody on polyethylene glycol-modified disposable screen-printed electrode as the detection platform, with polyclonal secondary anti-ENO1-tagged, gold nanoparticle (AuNP) congregates as electrochemical signal probes. The immunorecognition of the sample ENO1 by the congregated AuNP@antibody occurred on the surface of the electrodes; the electrochemical signal from the bound AuNP congregates was obtained after oxidizing them in 0.1 M HCl at 1.2 V for 120 s, followed by the reduction of AuCl(4-) in square wave voltammetry (SWV) mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 10(-8) to 10(-12) g/mL. This AuNP congregate-based assay provides an amplification approach for detecting ENO1 at trace levels, leading to a detection limit as low as 11.9 fg (equivalent to 5 microL of a 2.38 pg/mL solution).

Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum.

In this study, we demonstrated that the fiber-optic biosensor based on localized surface plasmon coupled fluorescence (LSPCF) is capable of detecting alpha-fetoprotein (AFP) in human serum. The sensitivity of LSPCF fiber-optic biosensor is not only enhanced but also the specific selectivity is improved since the fluorophores are excited by the localized surface plasmon with high efficiency. Experimentally, this fiber-optic biosensor is able to detect AFP concentration in phosphate buffered saline (PBS) solution from 0.1ng/mL to 100ng/mL whereas the linear relationship between the AFP concentrations and the fluorescence signals is shown. Furthermore, a linear response between the fluorescence signals and the concentrations of AFP in human serum from 2.33ng/mL to 143.74ng/mL is also obtained. As a result, the detection limit of the LSPCF fiber-optic biosensor on AFP detection is comparable with the conventional enzyme-linked immunosorbent assay (ELISA). Additionally, the LSPCF fiber-optic biosensor benefits on inexpensive, disposable and simpler optical geometry that can become a high efficient immunoassay comparable with the conventional ELISA and radioimmunoassay (RIA) clinically.

Differential-phase surface plasmon resonance biosensor.

In this paper, a novel differential-phase-sensitive surface plasmon resonance biosensor (DP-SPRB) is proposed and developed, in which a two-frequency laser is integrated with a differential amplifier in order to analytically convert the phase modulation into amplitude modulation. With the use of the conventional envelope detection technique, the differential phase is precisely decoded in real time in terms of the demodulated amplitude. In order to verify high detection sensitivity of the DP-SPRB, a sucrose-water solution and glycerin-water solution at low concentrations were both tested, and the experimental results confirm that the detection sensitivity on wt % concentration of the sucrose solution is 0.00001%. Moreover, the real-time monitoring mouse IgG/antimouse IgG interaction shows the minimum concentration of mouse IgG to be at 10 fg/mL. To our knowledge, this is the highest sensitivity ever measured by a surface plasmon resonance biosensor. However, because of the limited dynamic range of DP-SPRB, it can only apply to biomolecule interactions at extremely low concentration.

Discrimination of Breast Cancer by Measuring Prostate-Specific Antigen Levels in Women's Serum

Prostate-specific antigen (PSA) has been reported to be a potential biomarker of breast cancer. Serum PSA of normal women is around 1 pg/mL, which is usually undetectable by current assay methods; thus an ultrasensitive measurement of PSA expression in womens serum is necessary to distinguish normal from malignant breast diseases. To enhance the sensitivity of conventional immunoassay technology for the detection of PSA in sera, we adopted a localized surface plasmon coupled fluorescence fiber-optic biosensor, which combines a sandwich immunoassay with the localized surface plasmon technique. The concentration of total PSA (t-PSA) (from 0.1 to 1000 pg/mL) in phosphate-buffered saline solution and the normalized fluorescence signal exhibit a linear relationship where the correlation coefficient is 0.9574. In addition, the concentration of additional t-PSA in 10-fold-diluted healthly womens serum across a similar range was measured. The correlation coefficient for this measurement is 0.9142. In clinical serum samples, moreover, the experimental results of t-PSA detection show that both the mean value and median of normalized fluorescence signals in the breast cancer group (155.2 and 145.7, respectively) are higher than those in the noncancer group (46.6 and 37.1, respectively). We also examined the receiver operating characteristic curve for t-PSA, and the area under the curve (AUC) is estimated to be 0.9063, the AUC being used to measure the performance of a test to correctly identify diseased and nondiseased subjects.

Detection of swine-origin influenza A (H1N1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Abstract Swine-origin influenza A (H1N1) virus (S-OIV) was identified as a new reassortant strain of influenza A virus in April 2009 and led to an influenza pandemic. Accurate and timely diagnoses are crucial for the control of influenza disease. We developed a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB) which combines a sandwich immunoassay with the LSP technique using antibodies against the hemagglutinin (HA) proteins of S-OIVs. The detection limit of the LSPCF-FOB for recombinant S-OIV H1 protein detection was estimated at 13.9pg/mL, which is 103-fold better than that of conventional capture ELISA when using the same capture antibodies. For clinical S-OIV isolates measurement, meanwhile, the detection limit of the LSPCF-FOB platform was calculated to be 8.25×104 copies/mL, compared with 2.06×106 copies/mL using conventional capture ELISA. Furthermore, in comparison with the influenza A/B rapid test, the detection limit of the LSPCF-FOB for S-OIV was almost 50-fold in PBS solution and 25-fold lower in mimic solution, which used nasal mucosa from healthy donors as the diluent. The findings of this study therefore indicate that the high detection sensitivity and specificity of the LSPCF-FOB make it a potentially effective diagnostic tool for clinical S-OIV infection and this technique has the potential to be applied to the development of other clinical microbe detection platforms.

Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Abstract In order to enhance the sensitivity of conventional immunoassay technology for the detection of SARS coronavirus (SARS-CoV) nucleocapsid protein (N protein), we developed a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor that combines sandwich immunoassay with the LSP technique. Experimentally, a linear relationship between the fluorescence signal and the concentration of recombinant SARS-CoV N (GST-N) protein in buffer solution could be observed from 0.1pg/mL to 1ng/mL. In addition, the concentration of GST-N protein in diluted serum across a similar range could also be measured. The correlation coefficients (linear scale) for these two measurements were 0.9469 and 0.9624, respectively. In comparison with conventional enzyme linked immunosorbent assay (ELISA), the detection limit of the LSPCF fiber-optic biosensor for the GST-N protein was improved at least 104-fold using the same monoclonal antibodies. Therefore, the LSPCF fiber-optic biosensor shows an ability to detect very low concentration (∼1pg/mL) of SARS-CoV N protein in serum. The biosensor should help with the early diagnosis of SARS infection.

Detection of prostate-specific antigen with a paired surface plasma wave biosensor.

In this study, we demonstrated that an amplitude-sensitive paired surface plasma wave biosensor (PSPWB) is capable of real-time detection of prostate-specific antigen (PSA) in diluted human serum without labeling. Experimentally, the detection limit of PSPWB was 8.4 x 10(-9) refractive index unit (RIU) and the PSPWB could measure PSA in a phosphate buffered saline solution from 10 fg/mL ( approximately 300 aM) to 100 pg/mL ( approximately 3 pM) successfully, with demonstration of a linear relationship between PSA concentrations and surface plasmon resonance (SPR) signals. Therefore, results were obtained over a wide dynamic range 5 orders of magnitude for analyte concentration. In addition, the PSPWB successfully detected PSA in diluted human serum as well. These experimental results indicate that the PSPWB is capable of detection with high sensitivity over a wide range by using SPR-based biosensors and has a capability of detecting biological analytes in clinical sample without complicated operating procedures.

Rapid and highly sensitive method for influenza A (H1N1) virus detection.

In this study, we applied the developed paired surface plasma waves biosensor (PSPWB) in a dual-channel biosensor for rapid and sensitive detection of swine-origin influenza A (H1N1) virus (S-OIV). In conjunction with the amplitude ratio of the signal and the reference channel, the stability of the PSPWB system is significantly improved experimentally. The theoretical limit of detection (LOD) of the dual-channel PSPWB for S-OIV is 30 PFU/mL (PFU, plaque-forming unit), which was calculated from the fitting curve of the surface plasmon resonance signal with a S-OIV clinical isolate concentration in phosphate-buffered saline (PBS) over a range of 18-1.8 × 10(6) PFU/mL. The LOD is 2 orders of magnitude more sensitive than the commercial rapid influenza diagnostic test at worst and an order of magnitude less sensitive than real-time quantitative polymerase chain reaction (PCR) whose LOD for S-OIV in PBS was determined to be 3.5 PFU/mL in this experiment. Furthermore, under in vivo conditions, this experiment demonstrates that the assay successfully measured S-OIV at a concentration of 1.8 × 10(2) PFU/mL in mimic solution, which contained PBS-diluted normal human nasal mucosa. Most importantly, the assay time took less than 20 min. From the results, the dual-channel PSPWB potentially offers great opportunity in developing an alternative PCR-free diagnostic method for rapid, sensitive, and accurate detection of viral pathogens with epidemiological relevance in clinical samples by using an appropriate pathogen-specific antibody.

The utility of a high-throughput scanning biosensor in the detection of the pancreatic cancer marker ULBP2.

In this study, a novel high-throughput biosensor based on metal-enhanced fluorescence technique and harmonic intensity-modulated fluorescence technique was developed and demonstrated to be highly sensitive for the detection of a pancreatic cancer marker, UL16-binding protein 2 (ULBP2), in diluted serum. Experimentally, the biosensor is able to detect ULBP2 at 16-18 pg/mL in 1% BSA-PBS and in 10-fold-diluted human serum. Compared with the limit of detection (LOD) of the conventional enzyme-linked immunosorbent assay (ELISA) method, the LOD of the proposed biosensor for ULBP2 is significantly improved by 100-fold under the same conditions. In addition, the proposed method uses two identical polyclonal antibodies for the sandwich immunoassay, simplifying the experiment in terms of the reagents needed. Consequently, this biosensor is a cost-effective tool for clinical diagnosis. We believe that the proposed high-throughput biosensor has great potential to become a clinical diagnostic tool for the detection of a pancreatic cancer marker in the near future.

Binding Kinetics of Biomolecule Interaction at Ultralow Concentrations Based on Gold Nanoparticle Enhancement

Measuring the kinetic constants of protein-protein interactions at ultralow concentrations becomes critical in characterizing biospecific affinity, and exploring the feasibility of clinical diagnosis with respect to detection sensitivity, efficiency and accuracy. In this study, we propose a method that can calculate the binding constants of protein-protein interactions in sandwich assays at ultralow concentrations at the pg/mL level, using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). We discuss a two-compartment model to achieve reaction-limited kinetics under the stagnant conditions of the reaction chamber. The association rate constant, dissociation rate constant, and the equilibrium dissociation constant, that is, k(a), k(d), K(D), respectively, of the kinetics of binding between total prostate-specific antigen (t-PSA) and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that k(a), k(d), and K(D) have been measured at such a low concentration range in a complex sample such as human serum.