[Show abstract][Hide abstract] ABSTRACT: In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.
The Analyst 02/2012; 137(4):1013-9. DOI:10.1039/c2an15902c · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two-color DNA microarray platforms are widely used for determining differential amounts of target sequences in parallel between sample pairs. However, the fluorescence (or Forster) resonance energy transfer (FRET) between two fluorophores can potentially result in the distortions of the measured fluorescence signals. Here we assessed the influence of FRET on the two-color DNA microarray platform and developed a reliable and convenient method for the correction of FRET distortion. Compared to current methods of normalization based on the statistical analysis and the hypothesis that only a small part of target sequences are differentially presented between sample pairs, our FRET correction method can recover the undistorted signals by the compensation of fluorescence emission, without considering the number of target sequences differentially presented. The correction method was validated with samples at different target ratios and with microarrays spotted in different probe concentrations. We also applied the FRET correction method to gene expression profiling arrays, and the results show that FRET was present when the content of target sequence was beyond a threshold amount and that the process incorporating our FRET correction method can improve the reliability of the gene expression profiling microarray platform in comparison with the current process without FRET correction.
[Show abstract][Hide abstract] ABSTRACT: Fluorescence detection using two spectrally distinct fluorophores has long been used for the determination of the relative abundance of biomolecules, but overlap between the fluorescence spectra of each fluorophore can result in nonradiative Förster resonance energy transfer (FRET) and distorting the signals detected by fluorescence channels. Thus conventional methods for quantifying the relative abundance of fluorophores by fluorescence emission will not be accurate if FRET can occur. In this paper we report the development of a quantitative fluorescence correction method incorporating FRET to measure the relative abundance of fluorophores in dual-labeling experiments. The quantitative fluorescence correction method incorporating FRET is accurate, comprehensive, and convenient for the measurement of the relative abundance of fluorophores in dual-labeling experiments and can also correct the FRET distortion and provide accurate, quantitative, and convenient measurement of the hybridization efficiencies on microarrays.
[Show abstract][Hide abstract] ABSTRACT: In this paper, we developed a microsphere enhanced and label free high throughput molecular detection based on SPRI and microarray chips, and a self-built surface plasmon resonance imaging instrument was set. One label-free protein chip forming a 7× probe array was designed and fabricated using a commercial microarray robot spotter on chemical modified gold-coated glass slide. Antibody molecules were successfully detected in label-free and high-throughput method by using this chip. The detection signals on the chip was successfully enhanced by using microspheres with a diameter of 1 mum.
Proceedings of SPIE - The International Society for Optical Engineering 12/2008; DOI:10.1117/12.823373 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell migration is crucial in many physiological and pathological processes including embryonic development, immune response and cancer metastasis. Traditional methods for cell migration detection such as wound healing assay usually involve physical scraping of a cell monolayer followed by an optical observation of cell movement. However, these methods require hand-operation with low repeatability. Moreover, it's a qualitative observation not a quantitative measurement, which is hard to scale up to a high-throughput manner. In this article, a novel and reliable on-chip cell migration detection method integrating surface chemical modification of gold electrodes using self-assembled monolayers (SAMs) and real-time cellular impedance sensing is presented. The SAMs are used to inhibit cell adherence forming an area devoid of cells, which could effectively mimic wounds in a cell monolayer. After a DC electrical signal was applied, the SAMs were desorbed from the electrodes and cells started to migrate. The process of cell migration was monitored by real-time impedance sensing. This demonstrates the first occurrence of integrating cellular impedance sensing and wound-forming with SAMs, which makes cell migration assay being real-time, quantitative and fully automatic. We believe this method could be used for high-throughput anti-migratory drug screening and drug discovery.
Lab on a Chip 07/2008; 8(6):872-8. DOI:10.1039/b804130j · 5.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A surface plasmon resonance imaging (SPRI) system was developed for the discrimination of proteins on a gold surface. As a label-free and high-throughput technique, SPRI enables simultaneously monitoring of the biomolecular interactions at low concentrations. We used SPRI as a label-free and parallel method to detect different proteins based on protein microarray. Bovine Serum Albumin (BSA), Casein and Immunoglobulin G (IgG) were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6 x 6 matrix. These proteins can be discriminated directly by changing the incident angle of light. Excellent reproducibility for label-free detection of protein molecules was achieved. This SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.
Advances in Biomedical Photonics and Imaging - 6th International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2007); 06/2008
[Show abstract][Hide abstract] ABSTRACT: Biochips have been an advanced technology for biomedical applications since the end of the 20th century. Optical detection systems have been a very important tool in biochip analysis. Microscopes are often inadequate for high resolution and big view-area detection of microarray chips, thus some new optical instruments are required. In this work, a novel digital imaging scanning system with dark-field irradiation is developed for some biomedical applications for microarray chips, characterized by analyzing genes and proteins of clinical samples with high specific, parallel, and nanoliter samples. The novel optical system has a high numerical aperture (NA=0.72), a long working distance (wd>3.0 mm), an excellent contrast and signal-to-noise ratio, a high resolving power close to 3 mum, and an efficiency of collected fluorescence more than two-fold better than that of other commercial confocal biochip scanners. An edge overlap algorithm is proposed for the image restructure of free area detection and correcting scanning position errors to a precision of 1 pixel. A novel algorithm is explored for recognizing the target from the scanning images conveniently, removing noise, and producing the signal matrix of biochip analysis. The digital imaging scanning system is equally as good for the detection of enclosed biochips as it is for the detection of biological samples on a slide surface covered with a glass cover slip or in culture solution. The clinical bacteria identification and serum antibody detection of biochips are described.
[Show abstract][Hide abstract] ABSTRACT: A novel confocal optical system design and a dual laser confocal scanner have been developed to meet the requirements of highly sensitive detection of biomolecules on microarray chips, which is characterized by a long working distance (wd>3.0 mm), high numerical aperture (NA=0.72), and only 3 materials and 7 lenses used. This confocal optical system has a high scanning resolution, an excellent contrast and signal-to-noise ratio, and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners. The scanner is as equally good for the molecular imaging detection of enclosed biochips as for the detection of biological samples on a slide surface covered with a cover-slip glass. Some applications of gene and protein imagings using the dual laser confocal scanner are described.
International Journal of Biomedical Imaging 08/2007; 2007:79710. DOI:10.1155/2007/79710
[Show abstract][Hide abstract] ABSTRACT: Fluorescence resonance energy transfer (FRET) has found wide use in structural biology, biochemistry, and cell biology for measuring intra- and inter-molecular distances in the 1-10 nm range and for obtaining quantitative spatial and temporal information about the interaction of proteins, lipids, and DNA. The measurements of distances and interactions are based on the calculation of the fluorescence transfer efficiency using some algorithms to process the acquired images from several different filter sets. However, FRET measurements can suffer from several sources of distortion because of cross talk between donor and acceptor fluorophores. In this paper, we measured the FRET efficiency on glass coverslips using microarray technology and described an algorithm to analyze the FRET data obtained, which is corrected for the cross talk due to spectral overlap of donor and acceptor molecules. Measurement of the interaction of the donor and acceptor, which are mixed together or coupled to the respective 3'-end and 5'-end of a single-strand DNA are shown to document the accuracy of the approach, and allow one to estimate cross talk between the different filter units and to reveal the relationship of the FRET efficiencies of these two samples relative to the donor and acceptor concentrations.
Proceedings of SPIE - The International Society for Optical Engineering 05/2007; DOI:10.1117/12.741482 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surface plasmon resonance (SPR) technique is based on an optical measurement approach that is highly sensitive to the refractive index unit (RIU) of the sample on its analysis surface. Here, we demonstrate the direct detection of proteins and small molecules using an advanced SPR technology with a sensitivity that is as good as Fourier transform infrared (FTIR) spectroscopy. Some quantitative results are reported in this paper.
Proceedings of SPIE - The International Society for Optical Engineering 05/2007; DOI:10.1117/12.741483 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since microscope was invented in 17 century, optical instruments have continued to be an important tool for advanced investigations in biology and medicine. The step by step development of optical technology has also preceded the realization of macroscopic and microscopic biology, providing new advances that help better understanding of these fields. Even today it is necessary to build new optical devices to foster the advancement of frontier biological research, and for new applications in medical research. Here in this paper, we describe two new advanced optical systems for biological detection, which are already used in a series of new instrument products, such as the confocal scanner, the digital imaging scanning system.
Proceedings of SPIE - The International Society for Optical Engineering 05/2007; DOI:10.1117/12.741309 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microscopy is an important tool in biology and medicine, but it is often limited to high numerical aperture (NA) with a short working distance(wd), such as NA = 0.6 then wd < 1 mm. For imaging living cells in culture, a high numerical aperture and long working distance of optical imaging structure is required, and the common microscopy objective is no good here. In order to meet the single living cell imaging, a novel optical design has been performed in this paper, which is character of ultra-long working distance wd = 15 mm and high numerical aperture NA = 0.7. An optical imaging system with dual modes information of fluorescence and spectrum can be developed for the subcellular imaging of cells attached on surface of vessel or free-floating in culture.
[Show abstract][Hide abstract] ABSTRACT: Fluorescence resonance energy transfer (FRET) is a distance-sensitive energy transfer process which is widely used in probing molecular interaction in the 1-10 nm range. DNA microarray chip technique is a high throughput analysis method in molecular biology research. In this paper, we explored a novel detection and analysis method of DNA microarray hybridization using FRET technique. In our study, TMR dye labeled DNA oligomer was immobilized on the microarray chips and hybridized with complementary DNA oligomer labeled with Cy5 dye. We successfully detected and analysed the FRET signal of the hybridized DNA microarray. The variation of FRET signal intensity and the efficiency of FRET in response to the concentration of the sample were studied.