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EP-3802869-B1 - DEVICES AND METHOD FOR DETECTING AN AMPLIFICATION EVENT

EP3802869B1EP 3802869 B1EP3802869 B1EP 3802869B1EP-3802869-B1

Inventors

  • GEORGIOU, PANTELIS
  • MONIRI, Ahmad
  • MOSER, NICOLAS
  • RODRIGUEZ MANZANO, Jesus

Dates

Publication Date
20260513
Application Date
20190607

Claims (15)

  1. A method for detecting an amplification reaction in a solution containing a biological sample using an array of ion sensors, the amplification reaction being indicative of the presence of a nucleic acid, the method comprising: monitoring a signal from each respective sensor of the array of ion sensors; detecting a change in the signal from a first sensor of the array of ion sensors; comparing the signal from the first sensor with the signal of at least one neighbouring sensor, the at least one neighbouring sensor being proximate to the first sensor in the array, wherein comparing the signal from the first sensor with the signal of the at least one neighbouring sensor comprises calculating a correlation parameter between the signal of the first sensor and the signal of the at least one neighbouring sensor; and determining, based on the comparing, that an amplification event has occurred in the solution in the vicinity of the first sensor.
  2. The method of claim 1, wherein the amplification event comprises a single molecule amplification event.
  3. The method of claim 1 or claim 2, further comprising detecting a plurality of amplification events, and monitoring the events as they occur to obtain a frequency value indicative of the rate of the reaction.
  4. The method of claim 3, wherein the amplification events are single molecule events and monitoring the events as they occur comprises counting the single molecule events.
  5. The method of any preceding claim, wherein detecting a change in the signal from the first sensor comprises comparing the received signal from the first sensor to a previously received signal from the first sensor and determining that a change in signal value between the received signal and previously received signal is greater than a threshold value.
  6. The method of any preceding claim, wherein comparing the signals comprises monitoring a degree of similarity between the signals received from the first sensor and the signals received from the neighbouring sensor.
  7. The method of claim 6, wherein determining that an amplification event has occurred comprises determining that the degree of similarity between the signals received from the first sensor and the signals received from the neighbouring sensor is greater than a similarity threshold.
  8. The method of any preceding claim, wherein the method is performed during a nucleic acid amplification reaction.
  9. The method of claim 8, wherein the nucleic acid amplification reaction is an isothermal reaction, and optionally wherein the reaction is a LAMP reaction.
  10. The method of any preceding claim, wherein the biological sample is at least one of a DNA, RNA or protein sample.
  11. The method of any preceding claim, wherein the ion sensors are any of ISFET sensors, pH sensors, or chemically sensitive sensors.
  12. The method of any preceding claim, wherein the method is a computer implemented method, and optionally wherein the method is carried out using an algorithm created using a machine learning technique.
  13. An apparatus comprising an array of ion sensors, a processor and a memory, the memory comprising instructions which, when executed by a processor, cause the processor to carry out the method of any preceding claim.
  14. A computer-readable medium comprising instructions which, when executed by a processor, cause the processor to carry out the method of any one of claims 1 to 12.
  15. A method of diagnosing a subject using the method of any of claims 1 to 12, the method comprising: bringing the solution containing a biological sample into contact with the array of ion sensors; determining that an amplification event has occurred in the vicinity of the first sensor, the amplification event being indicative of the presence of a particular pathogen; and determining that the patient has a particular disease based on the presence of the pathogen.

Description

This disclosure relates to detecting an amplification event, and in particular to detecting an amplification reaction in a biological sample using an array of ISFET sensors. Background The importance of fast, cheap, robust and quantitative detection of pathogens at the point-of-need cannot be stressed enough. However, detection of spatio-temporal chemical interactions at the molecular level, including nucleotide incorporation during nucleic acid amplification reactions and sequencing or primer-nucleic acid interactions, in real-time is not possible using current technologies without the use of high-end, expensive and bulky instruments. Enabling these capabilities would provide fundamental insights on chemical interactions and the kinetics of biological and chemical reactions at the molecular level, such as DNA replication, DNA transcription, RNA translation or antibody-antigen binding events, and would lead to the development of more efficient detection chemistries and diagnostic methods. There are three main classes of diagnostic methods for detection and identification of pathogens: classical microbiology techniques (such as microscopy and cultivation), protein-based (such as antigen-antibody interactions) and nucleic acid-based (such as sequencing, polymerase chain reaction and microarrays) methods. Typically, classical microbiological methods have unacceptably long cycle times and depend on visual observation. Protein-based approaches are cheap, fast, and small; however the output is qualitative rather than quantitative, and a high concentration of pathogen in a given sample is required. By contrast, current nucleic acid-based approaches have a quantitative output, i.e. return a verdict of either present or not present, and can detect relatively low concentrations of pathogen in a given sample. However, current techniques are expensive, slow, and require large optical equipment to perform. DNA amplification, the process of replicating DNA from one original DNA molecule, is used to amplify a single or a few copies of a segment of DNA generating thousands to millions of copies of a particular DNA sequence and can be used to determine whether a sample of human fluid or tissue contains DNA or RNA of a pathogen (such as viruses, bacteria, fungi or protozoa). The basic premise is that the DNA amplification is allowed if and only if the target pathogen exists. Following this, the DNA amplification is monitored. For instance, in traditional methods such as real-time polymerase chain reaction (PCR) each time a new amplicon is produced, a fluorescent molecule is released. Hence, the release of this fluorescent molecule is an indication of the presence of a pathogen in the sample. It is also possible to monitor the pH of the chemical solution because during DNA amplification, each time a nucleotide is incorporated into the new DNA strand, Hydrogen ions are released which cause a change in the pH (pH = -log10 [H+], where H+ is the concentration of Hydrogen ions or protons). The chemistry is summarised in the below equation where α is an integer constant. DNA+reactants−−>2⋅DNA+α⋅ProtonH++products If DNA amplification is triggered (i.e. the pathogen is present in the sample) then the reaction is defined as positive, otherwise, the reaction is described as negative. A high-level description of how pH-based DNA detection is typically performed is illustrated in Figure 1a and summarised in the following steps: 1. Chemical solution consisting of sample and other necessary chemicals is prepared.2. Amplification reagents associated with a specific pathogen is added to the solution. This consists of a primer, a sequence of bases, that complements the target DNA.3. Depending on the method of DNA detection, the chemical solution may be heated.4. Amplification is triggered if the primer complements the DNA in the sample.5. DNA amplification is monitored; for instance, through fluorescence or pH. Assuming no noise exists in the system, a typical output profile for DNA detection is shown in Figure 1b. This figure includes a typical profile for a positive and a negative reaction. The graph shows time on the x-axis, and pH (or fluorescence) on the y-axis. The graph is split into three 'stages' representing the expected profile for DNA amplification. At stage I) the reactants have not found each other yet. At stage II) amplification is taking place. At stage III) the reaction has saturated. The 'time to positive', tp, is defined as the time from the beginning of the reaction until a positive determination that the DNA is amplifying. Since the threshold is arbitrary, in examples used herein tp may be taken as the time for half of the amplification to complete. Traditional methods of nucleic acid-based detection use optical mechanisms based on fluorescence labelling that require large and costly equipment. Typically, this equipment makes such techniques unsuitable for point-of-care diagnostics. Polymerase chain reaction (PCR), is the