A Novel Colorimetric Array Sensor For The Detection Of Cellular Metabolic Process

Cell metabolism is intimately associated with cellular life activities, and it gives expression to the cellular physiological and pathological states accurately to some extent. Comparing with genes or proteins, cellular metabolites play an important role in cell signal transduction, intracellular energy transfer and other life processes in the metabolic level. The regulation of cellular metabolites has special influence on life processes. Thus, the qualitative and quantitative detection of biological molecules, as well as the studies of finding relative relationship between cellular metabolite changes and physiological or pathological states, plays a critical role to medical diagnosis. Currently, traditional large-scale equipment testing method for the identification and quantification of biological molecules and biological systems is commonly used, although it ensures the accuracy of detection, the high cost and low detection efficiency are tedious and cumbersome. The newly developted electronic noses/ tongues are well perfected in relevant theorys and fabricated methods, the difficulties on selectivity and stability of sensitive materials limites the wildly application of those sensors. In this study, based on cross-reactive mechanism, a novel visualized sensor array is developed for rapid and sensitive identification of biological molecules, even biological systems.By stimulating mammalian olfactory/ gustatory sense, visualized sensor arrays utilize variety specific and sensitive material as sensitive unit, treat target mixtures as an individual sample to extract features as unique fingerprints, and then recognize and identify target mixtures with pattern recognition algorithm. Thus, the visualized sensor arrays have unique advantages in the recognition of mixtures. Recently, because of the good biocompatibility and sensitive performance, porphyrin and its derivatives are employed as sensitive material for the visualized sensor array. The colorimetric sensor array has advantages in high sensitivity, good stability, and it is easy-to-modification and so on. In this study, with porphyrin and its derivatives as sensitive materials, a colorimetric sensor array was fabricated for the recognition and identification of amino acids, proteins and cellular metabolism detection, as well as the specific recognition to metabolites of normal and pathological cell in different life cycle can be achieved, and it is expected to provide an effective platform for drugs screening in vitro. Thus, the works presented here can be summarized as follows:1. The preparation and selection of sensitive material To detect cellular metabolites, amino acids were assumed as target molecules. Water- soluble porphyrin substituted with sulfonic groups (PS1 and PS4) and metal porphyrin derivatives (P1-P5) were synthesized based on Adler method. The interactive manners and the UV-visible spectroscopic results between dyes (e.g. porphyrin, porphyrin derivatives and indicator dyes) and amino acids suggested that those dyes can combine with amino acids in varying degrees. They had a certain affinity and selectivity to the molecules of amino acid, resulting in various specific pattern models for signal processing. Thus, a visualized sensor array with 36 sensitive elements was fabricated using similar approaches. Based on cross- reactive mechanism, each sensitive element provided individual signal to target biological molecules. Thus, it realized the recognition and identification of biological molecules, even the cellular life cycles.2. The design and fabrication of colorimetric sensor array systemThe detail of the fabricated processes was availabled for the design and construct of colorimetric sensor array, including hardware design, software design and the sensor array design. The process of image acquisition before and after reaction was availabled in hardware design. And the processing of images and visualization of data were realized by software design. The colorimetric sensor array system was specially developed using sol-gel method. Briefly, tetraethyl orthosilicate (TEOS), ultra-pure water, hydrochloric acid (1 mol/L), ethanol and indicator solution (porphyrin, porphyrin derivatives and indicator dyes) were mixed with a volume ratio of 25:4:3:15:10. A quartz capillary was used to deliver sol-gel onto the surface of a porous hydrophobic membrane. Thus a 6×6 colorimetric sensor array was fabricated for the detection of biological molecules and biological system, layed the foundation for the construction of artificial tongue system.3. Important biomolecules and biosystems, e.g. amino acids, carbohydrate compounds and proteins, were detected by the colorimetric sensor array system.Amino acids, carbohydrate compounds and proteins are the basic material for living organisms, as well as the molecular basis for cellular metabolism. Ten kinds of amino acids, nine carbohydrate compounds and mixtures, senven pure proteins and protein mixtures, and three thermal denatured proteins were identified by the developed colorimetric sensor array system. And the unique fingerprints of those biological molecules were obtained. Combination of UV-visible spectroscopy and density functional theory, the interaction between PS1 and amino acids was analyzed by quantum chemical theory and experimental results, which suggested the structure of amino acid residues had great influence on interactions between porphyrin molecules and amino acid. The RGB values of the colorimetric sensor array were analyzed by pattern recognition method, including cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (LDA). The results suggested that different amino acids can be easily distinguished by the developed colorimetric sensor array. It can even reflect the chemical property difference of the different amino acids. Meanwhile, the developed visualized sensor array system is an excellent sensing platform for the identification and quantitative analysis of carbohydrate compounds and protein samples, and the unique pattern of colorific change may be due to the change of conformation and their characteristic.4. Colorimetric sensor array for metabolic fingerprint of endothelial cell growth cycleUsing 16 chemically responsive dyes (porphyrin, porphyrin derivatives and indicator dyes) as sensor elements, the developed sensor array showed a unique pattern of colorific change to the metabolist of human umbilical vein endothelial cells (HUVEC) cultured 1-10d. The digital data library generated was analyzed with statistical and chemometric methods, including PCA and HCA. Comparing with traditional MTT colorimetry, digital imaging of the dye array before and after immersion provides a color change profile as a unique fingerprint for each cellular metabolic liquid cultured from 1 to 10 days. Facile identification of all the cellular metabolic liquids was readily achieved by using comparison of the color change profiles or a PCA score plot. The UV-visible spectra also indicated that the responsive color change profiles can significantly discriminate the cellular metabolic samples. These results demonstrated the potential application of this developed colorimetric sensor arrays for monitoring cellular growth cycle, cell recognition, cell pathology, pharmacology and genetaceutical screening.5. The identification of normal human synovial (NHS) and rheumatoid arthritis human synovial (RA) cells based on microfluidic technologyThe preparation of microfluidic chip was introduced in detail, and its effectes on cellular segregation and culture were evaluated. The 16 elements visualized sensor arrays were used to detect the metabolists of NHS and RA cultured from 1 to 9 days, thus color change profiles as unique fingerprints for each cell were achieved. HCA and PCA analysis showed that the colorimetric sensor array can distinguish the different stages of the cells growth. The 57 samples tested by LDA analysis had the overall accuracy rate as 94% for cellular metabolic liquid cultured from 1 to 9 days, indicating that the colorimetric sensor array can significantly discriminate the cellular metabolic samples, even trace metabolites changes. In consequently, the fabricated colorimetric sensor array system not only provided a good research platform for the studies on pathogenesis of rheumatoid arthritis, but also had potential application on the relevant anti-arthritis drugs, anti-angiogenesis drug screening.