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CA-3129896-C - SELECTIVE OPTICAL DETECTION OF ORGANIC ANALYTES IN LIQUIDS

CA3129896CCA 3129896 CCA3129896 CCA 3129896CCA-3129896-C

Abstract

Present invention relates to a method and a device for detection and quantification of various organic analytes in liquid sample and an assay substrate for providing analyte measurements. In said method is implemented by using specific interaction of organic analytes with selective binding sites immobilized on the multi-layer assay substrate with further detection of such interaction with a device based on inducing and recording the fluorescence of such substrate for bio-chemical, genetic and environmental analyses.

Inventors

  • Sergei Babitshenko
  • JAAK JARV
  • Aleksei Kuznetsov
  • Anton Mastitski

Assignees

  • QANIKDX OU

Dates

Publication Date
20260505
Application Date
20200120
Priority Date
20190211

Claims (20)

  1. 36 WHAT IS CLAIMED IS: 1. An assay substrate comprising: a first component comprising a sensor molecule labeled with a quantum dot, the quantum dot immobilized to an assay substrate surface with a first linker, the sensor molecule having a specific binding site for an organic analyte, the sensor molecule labeled with the quantum dot in a position that has no effect on the organic analyte binding the specific binding site; a second component comprising a chemical analogue of the organic analyte, the chemical analogue labeled with a fluorescent dye, the chemical analogue linked to the quantum dot with a second linker having a length exceeding Forster radius, and the chemical analogue reversibly binding the specific binding site of the sensor molecule of the first component; the quantum dot having a first fluorescence emission spectrum that excites fluorescence of the fluorescent dye and the fluorescent dye having a second fluorescence emission spectrum distinguished from the first fluorescence spectrum of the quantum dot.
  2. 2. The assay substrate of claim 1, wherein the second linker is sized to prevent binding of the chemical analogue with a specific binding site of a neighbouring first component unlinked to the chemical analogue.
  3. 3. The assay substrate of claim 1 or 2, wherein the sensor molecule is labeled with the quantum dot by a third linker linking the sensor molecule to the quantum dot, a length of the third linker being shorter than the Forster radius and being shorter than the length of the second linker.
  4. 4. The assay substrate of any one of claims 1-3, further comprising an assay substrate compartment for applying the liquid sample to the assay substrate surface; the assay substrate compartment limits a volume of the liquid sample applied onto the assay substrate surface, and limits a thickness of interaction surface layer of the liquid sample applied onto the assay substrate according to the Forster radius.
  5. 5. The assay substrate of any one of claims 1-4, wherein the first linker being a bi-polar linker comprising a first binding group for specific binding of the quantum dot and a second binding group for specific binding of the assay substrate surface.
  6. 6. The assay substrate of any one of claims 1-5, wherein the assay substrate surface is solid, chemically stable, and carries chemically active groups covalently linked to the first linker. Date rei,me/Date Received 2024-01-19 37
  7. 7. A system comprising: the assay substrate of any one of claims 1-6; a light source configured to emit a specific spectrum to excite fluorescence of the quantum dot; an opto-electronic detector configured to detect fluorescence of the fluorescent dye and generate a signal corresponding to fluorescence intensity; a controller configured to record the signal from the opto-electronic detector and determine presence of the organic analyte based on a decrease of the detected fluorescence.
  8. 8. The system of claim 7, wherein the controller is configured to record the signal generated by the opto-electronic detector in time to record a time curve of the detected fluorescence to determine the concentration of the organic analyte.
  9. 9. The system of claim 8, wherein the controller is configured to derive concentration of an analyte based on determining a relative decrease of recorded fluorescence in time from its initial value.
  10. 10. The system of any one of claims 7-9, wherein the controller is configured to process a detected fluorescence having multichannel characteristics.
  11. 11. The system of any one of claims 7-9, wherein the controller is configured to process a detected fluorescence having multispectral characteristics.
  12. 12. A method for detection of an organic analyte in a liquid sample, the method comprising: providing an assay substrate comprising a first component and a second component; the first component comprising a sensor molecule labeled with a quantum dot, the quantum dot immobilized to an assay substrate surface with a first linker, the sensor molecule having a specific binding site for an organic analyte, the sensor molecule labeled with the quantum dot in a position that has no effect on the organic analyte binding the specific binding site; the second component comprising a chemical analogue of the organic analyte, the chemical analogue labeled with a fluorescent dye, the chemical analogue linked to the quantum dot with a second linker having a length exceeding Forster radius, and the chemical analogue reversibly binding the specific binding site of the sensor molecule of the first component; Date rei,me/Date Received 2024-01-19 38 applying the liquid sample to the assay substrate; illuminating the assay substrate to excite fluorescence of the quantum dot; detecting fluorescence of the fluorescent dye; determining presence of the organic analyte by detecting a decrease in fluorescence of the fluorescent dye, due to the organic analyte displacing the chemical analogue from the specific binding site and subsiding a fluorescence resonance energy transfer (FRET) effect between the quantum dot and the fluorescent dye.
  13. 13. The method of claim 12, further comprising recording a time curve of the detected fluorescence.
  14. 14. The method of claim 13, further comprising determining a concentration of the organic analyte in the liquid sample based on the recorded time curve.
  15. 15. The method of claim 14, wherein determining the concentration is based on determining a relative decrease of recorded fluorescence in time from its initial value.
  16. 16. The method of any one of claims 13-15, wherein recording of the time curve comprises repeated cycles of excitation of fluorescence of the quantum dot and detection of fluorescence of the fluorescent dye and recording a value of the detected fluorescence at predetermined time intervals.
  17. 17. The method of any one of claims 13-16, wherein applying the liquid sample to the assay substrate occurs after initiating recording of the time curve.
  18. 18. The method of claim 12 or 13, further comprising determining an amount of the organic analyte in the liquid sample by measuring the decrease in the detected fluorescence of the fluorescent dye, the degree of the measured decrease corresponding to the amount of the organic analyte in the liquid sample.
  19. 19. The method of any one of claims 12-18, wherein the determining presence of the organic analyte occurs in a thin layer of the liquid sample, the thickness of an interaction surface layer of the liquid sample on the assay substrate being limited according to the Forster radius.
  20. 20. The method of any one of claims 12-19, wherein preparing the assay substrate comprises reversibly binding the chemical analogue to the specific binding site of the sensor molecule of the first component to position the fluorescent dye close to the quantum dot at a distance shorter than the Forster radius to enable the fluorescence resonance energy transfer (FRET) effect between the quantum dot and the fluorescent dye. Date rei,me/Date Received 2024-01-19

Description

1 Description Selective optical detection of organic analytes in liquids Technical Field [0001] The invention relates to a method, system and/or device for detection of an organic analyte in a liquid, and more specifically to a method system and/or device for detection of an organic analyte in a liquid by using fluorescence to detect an interaction with a corresponding immobilized binding site. Background Art [0002] Determination of small organic molecules in various natural liquid media is one of the most important and demanding tasks of bio-chemical, genetic, and environmental analyses, and different classical analytical methods are used to address these applications. [0003] Express spectrometric -methods are widely used in environmental monitoring of water pollution, in scientific research and medical diagnostics. Various types of equipment have been developed, including portable devices for field analysis (Long et al. 2013). In these devices, in case of fluorescence spectroscopy, the known volume of sample is processed and placed in a test tube and fluorescence of this sample is measured at specific excitation wavelength. This is a simple and fast method of analysis, if the sample is characterized by sufficiently different excitation and emission spectral bands. Some complications can be connected with the need to calibrate the device for each type of sample to take into consideration the influence of additional emitters or quenchers of fluorescence signal, as well as the influence of opalescence caused by solid particles present in analysed samples. These factors complicate the analytical procedure and may cause the measurement error. [0004] Such complications can be avoided if analyte is isolated from the liquid sample by the method of capillary electrophoresis and thereafter is detected with appropriate detector system by measuring, for example, UV spectrum or by using some another analytical method. This approach provides high detection sensitivity with application of portable devices (Lara et al. 2016). On the other hand, this method needs exact 2 determination of the electrophoretic mobility of the analyte in different types of samples, as this parameter can be dependent on sample type. Moreover, properties of the capillary used for electrophoresis may also depend on sample properties and its variation in time. Therefore replacement of capillary and re-calibration of the device is necessary to do on regular basis. Finally, this method needs additional check that the output signal is caused only by the analyte and does not include signal generated by other components of similar mobility. Validation of these results can be done by using other analytical methods, which are free from these complications. [0005] Among these methods the tandem technologies GC/MS, HPLC/MS or LCMS/MS (Buchberger 201 0; Farre et al. 2007; Petrovic et al. 2010) have central position. Although different usable devices have been developed, these methods cannot be used without sample preparation and require trained staff. Most importantly, these devices have remained expensive, especially if real-time analytical runs are considered (Staples et al. 2001 ). [0006] Electrochemical sensors are widely used in portable devices, which measure electric conductivity of the sample during some specific reaction taking place in the presence of analyte. These measurements can be made with great accuracy and the size of devices may be significantly reduced due to the possibility to use miniature chips with printed electrodes (Couto et al. 2015). The disadvantage of these sensors is connected with the detection procedure, where formation or disappearance of ionic compounds is measured in some set of consecutive reactions that occur in the presence of analyte, as each step of this reaction cascade may be influenced by the presence of impurities, properties of the reaction medium or temperature. All these factors contribute into uncertainty of the measurement, especially in the field conditions, and therefore these devices are mostly used for purposes of qualitative analysis. [0007] More recently synthetic oligonucleotides, named aptamers, were proposed for binding analyte molecules. Aptamers form spatial molecular structure that specifically recognizes the whole analyte molecule or some part of its 3 structure. Although discovery of aptamers has significantly widened analytical possibilities, based on creation of analytical chips with coatings sensitive to a particular analyte, still the absence of efficient and reliable detection methods has hindered development of cheap and efficient analytical and diagnostic devices. [0008] Aptamers are widely used in combination with Surface Plasmon Resonance (SPR) technique (Kodoyianni 2011 ). In this case aptamers are immobilized on the chip surface and the complex formation process is recorded by monitoring the change of the molecular mass of this complex. Although this approach seems to be rather gene