Microarray Reference Spots for Hybridization Failure Detection
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Solution Overview
Problem
Existing microarray detection methods face challenges in objectively determining performance degradation, insufficient washing, hybridization failure, and defective target polynucleotides, leading to ambiguous decision errors and the inability to process large samples automatically.
Innovation Solution
A microarray with both detection spots and reference spots, where the latter contains immobilized probe polynucleotides of multiple types, allows for fluorescence measurement to assess hybridization reliability and spot positioning, enabling reliable microarray operation and automatic data processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a microarray with only detection spots is used, then the device complexity is low, but the reliability of detection is insufficient due to inability to objectively determine performance degradation and hybridization failure
Solution Approach 1:
The microarray is segmented into two functional regions: detection spots for sample analysis and reference spots for quality control. This segmentation allows independent evaluation of hybridization reliability without complicating the overall device structure, as the reference spots serve a distinct validation function
Solution Approach 2:
Reference spots act as an intermediary control mechanism that mediates between the detection spots and the evaluation system. By providing a known hybridization target in the reference spots, the system can objectively assess whether hybridization failures or performance degradation occurred, eliminating the need for complex external validation procedures
2Productivity
If manual determination of decision errors is performed, then the measurement precision can be maintained, but the productivity is low due to inability to process large number of samples automatically
Solution Approach 1:
The microarray performs self-validation through the reference spots, which automatically indicate whether hybridization conditions were appropriate. This self-service mechanism eliminates the need for manual assessment of each detection spot, enabling automatic processing of large sample numbers while maintaining decision accuracy through objective reference-based evaluation
Solution Approach 2:
The reference spots provide immediate feedback on hybridization success or failure. This feedback mechanism allows the system to automatically identify and exclude unreliable detection results, maintaining measurement precision while enabling high-throughput automatic processing by eliminating subjective manual evaluation
3Reliability
If additional reagents are added to improve detection reliability, then the reliability of hybridization reaction determination improves, but the loss of substance increases
Solution Approach 1:
The reference spots utilize the same fluorescently labeled target polynucleotides already present in the sample, serving multiple functions: both as detection targets for sample analysis and as internal controls for reliability assessment. This multi-functionality eliminates the need for additional reagents while maintaining high detection reliability
Solution Approach 2:
The system uses the sample's own fluorescently labeled target polynucleotides to validate hybridization reliability through the reference spots. This self-service approach requires no additional reagents, as the existing sample components serve dual purposes: detection and quality control, thereby minimizing reagent consumption while ensuring reliable results
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach ensures reliable hybridization reaction determination and suppresses decision errors, enabling high-reliability automatic microarray measurement without additional reagents, improving the detection of hybridization failures and performance degradation.
Implementation Method 1
a fluorescent labeled target polynucleotide was brought into contact with the microarray and a hybridization reaction is caused to proceed... measuring the fluorescence of the reference spot
Implementation Method 2
The target polynucleotide, which has a complementary nucleotide sequence to the probe polynucleotide, complementarily hybridizes with the probe polynucleotide
Data Source
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AI summary
An object of the present invention is to provide means for objectively determining hybridization failure in addition to performance degradation, insufficient washing and the like in each microarray, and more specifically the present invention relates to a method for detecting the hybridization between a probe polynucleotide and a target polynucleotide by using a microarray, including 1) a step of bringing a fluorescent labeled target polynucleotide into contact with a microarray having not only a plurality of detection spots in which a plurality of types of probe polynucleotides are each immobilized but also at least one reference spot in which at least two types of probe polynucleotides immobilized in the detection spots are immobilized, 2) a step of removing an unreacted target polynucleotide by washing the microarray, 3) a step of measuring fluorescence of the reference spot and determining that if a predetermined value is satisfied, measurement can be made, and 4) a step of measuring fluorescence of each of the detection spots in which the probe polynucleotides are immobilized, if it is determined that measurement can be made.