The Construction Of Biomolecules Coding Assay: A General High Throughput Detection Technique

To detect biomolecules and their concentrations within a sample is very important in the fields of biological research and clinical diagnosis. Unfortunately, too many tests are needed for the detection of some target molecules (TMs) before the appearance of high throughput (HT) detection methods. This greatly decreases the work efficiency, and more frequently the available tests are limited because the amounts of samples are scare.Biochips are developed based on the parallel detection idea and it can detect thousands of TMs within a sample in a single test. But the sensitivity and reproducibility of biochips are not satisfactory for routine clinical usage partly because of the steric effect caused by the solid-liquid-reaction between biochips and samples. The xMAP, another high throughput detection method invented by LUMINEX Corporation, promotes the reproducibility significantly by adopting a suspension hybridization mode. But the xMAP can hardly detect over 100 kinds of TMs in a single test. Moreover, the xMAP needs exquisite manufacturing process.Here we Here we constructed another high throughput detection technique completely different biochips and xMAP. This novel HT technique was named Biomolecules Coding Assay or BMCA for short. The BMCA based on the ideal of coding the Biomolecular Recognization Elements (BREs) which can bind TMs specifically to different BRE-CM by labeled with variant Coding Molecules (CMs), which make the BRE-CMs in the detection mixtures can be resolved by their difference.To verify this novel technique and explore the practicable protocol, the mini model of BMCA, the quantization method, the model of detection proteins by BMCA and the model of detection nucleic figments were studied. Based on the results of these studies, it can be concluded that:1. A classic BMCA protocol is(1) To Code BREs and make BMCA detection reagents.The BREs are changed to BRE-CMs when labeled with variant Coding Molecules (CMs), and BRE-CMs are mixed with certain concentration and some necessary buffers to make a detection reagent. In the BMCA detection reagent, some BRE-CMs which can bind the TMs (tBRE-CM) specifically and at least a reference BRE-CM (rBRE-CM) which can bind nothing in the BMCA detection system should be contained.(2) To performance the binding reaction between BMCA detection reagents and the sample. The detection reagent is mixed with a sample and some necessary buffer to accomplish binding reaction between TMs and tBRE-CMs. The corresponding tBRE-CMs will be consumed to form TM-BRE-CMs in the reaction.(3) To resolve the BRE-CMs in the BMCA reaction mixtures. The mixture of detection reagent and sample are analyzed by separation methods, and the BRE-CMs or TM-BRE-CMs in it will be resolved and quantitated.(4) To decide the concentrations of each TM in the detected sample. The concentrations of each TM in the detected sample can be calculated out by the consumption of the corresponding tBRE-CM, detail methods will be descripted in the following paragraphs.2. The quantitation equation of BMCA isWhere C_TMis the concentration of TM, k is the actual binding ratio of TM and BRE-TM, C_tBRE-CM is the concentration of tBRE-CM in the detection reagent, rR_{tBRE-CM/rBRE-CM} is the ratio of tBRE-CM/rBRE-CM in BMCA sample blank test, tR_{tBRE-CM/rBRE-CM} is the ratio of tBRE-CM/rBRE-CM in BMCA detection test.There are 2 exceptions of this equation.First, when the difference of rR_{tBRE-CM/rBRE-CM} and tR_{tBRE-CM/rBRE-CM} less than 3 times of the standard deviation ( rSD) of rR_{tBRE-CM/rBRE-CM}, the TMs should be seen as lower than the minimum detection level of BMCA, which isWhere C min is the minimum detection limit of BMCA, V R is the volume of detection reagent used in a BMCA, VS is the volume of the sample used in a BMCA.Second, When the value of rR_{tBRE-CM/rBRE-CM}-tR_{tBRE-CM/rBRE-CM}≤0,the concentration of the TM should be seen as greater than the maximum detection level of BMCA, which is3. The dsDNA may be the best CM of BMCA.The dsDNA meet all the needs of standards for a suitable CM, which is(1) rich polymorphism;(2) easy to be labled on the TM;(3) taking no effect on the bindings characteristic of BREs;(4) enough stability;(5) binding nothing in the BMCA detection system.4. BMCA has much higher throughput than xMAP.The throughput of BMCA is decided by both the polymorphism of CM characters and the identification ability of separation methods adopted. If taking dsDNA as CMs, their molecular weight can be varied with the stepping of about 660 Daltons to infinitude in theory. The polyacrylamide gel electrophoresis (PAGE) can resolve dsDNA with 1 bp variance. So the throughput of BMCA is infinite in theory. However, the length of dsDNA which can be detected by the separated methods is limited. Therefore, the throughput of BMCA is finite. For these reasons, it's hard to tell an accurate number about the throughput of BMCA. But it's affirmative that BMCA have much higher throughput than xMAP.5. The sensitivity and repeatability of BMCA is good.The minimum detection limit of BMCA is proportional to the concentration of tBRE-CM in the detection reagent and the standard deviation of the separation method adopted by the BMCA. This suggests that it's helpful to improve sensitivity of BMCA by reducing the concentration of tBRE-CM near to the minimum detection limit of the adopted separation method and advancing the repeatability of the adopted separation method. This also clues that the sensitivity of BMCA may be lower than the minimum detection limit of the adopted separation method. It is reported that the minimum detection limit of electrophoresis method and stratography is about 10-11g, so the sensitivity of BMCA is supposed as 10-11g or lower which can satisfy the clinical diagnosis and a majority of biological research.The BMCA is performed in a pure liquid environment, and the effect of stereospecific blockade is avoided. By taking the ratio of tBRE-CM and rBRE-CM, the sampling error will be decreased at maximum degree. So the repeatability of BMCA is decided by the separation methods adopted. Because the electrophoresis method and stratography are widely used in clinical diagnosis and researches, their repeatability are acceptable.6. There is no specificity difference between BMCA and other biomolecules detection methods.The specificity of BMCA is decided by the specificity of BREs which is widely confirmed by immunology and molecular biology. A suitable CM hardly influences the specificity of TMs and BREs, so there is no need to worry about specificity of BMCA.7. TroubleshootingIt was found that the substance in the detection sample can affect the concentrations of BRE-CMs if it has the same chemical nature as the CM adopted in BMCA.When labelling another molecules such as fluorescein on the CM as a reporter, this disadvantages will be overcome.In conclusion, BMCA is a sensitive, specific, and accurate detection method with high throughput. No special equipment or complex process is needed in BMCA. Just using the electrophoresis apparatus or chromatograph which can be available in most laboratory, after producing some dsDNA fragments and labeling them to the BREs, the researchers can detect any biomolecules with high efficiency, reliable results and low cost.