WO2017072799A1

WO2017072799A1 - A system and a method for monitoring nucleic acid amplification reaction

Info

Publication number: WO2017072799A1
Application number: PCT/IN2016/050375
Authority: WO
Inventors: Gempuraj PURUSHOTHAMAN; Shaheed Shajahan MOHAMMED
Original assignee: M/S. Purius Nanosystems Private Limited
Priority date: 2015-10-30
Filing date: 2016-10-30
Publication date: 2017-05-04

Abstract

A system and a method for monitoring nucleic acid amplification is disclosed. The system comprising a test cartridge, a DAC biasing voltage circuit and a controlled thermal element. The test cartridge comprising a plurality of reaction chambers, wherein each reaction chamber encloses an electrolyte insulator semiconductor capacitor (EISCAP) sensor, said reaction chamber having a provision to hold a liquid sample comprising a nucleic acid amplification reaction mixture necessary for amplification of a target nucleic acid sequence if the target nucleic acid sequence is present. The liquid sample in the reaction chamber is sealed with a lid or a cap. The controlled thermal element is placed in close proximity to the reaction chamber for subjecting liquid sample to predefined temperatures for predefined time durations. The DAC biasing voltage circuit applies a bias voltage across the EISCAP sensor resulting in change of capacitance of EISCAP sensor, said capacitance value is plotted against said bias voltage to obtain multiple C-V curves during the reaction in real time.

Description

FIELD OF THE INVENTION

This invention relates to real-time monitoring of nucleic acid amplification reaction. BACKGROUND OF THE INVENTION

DNA (Deoxyribonucleic Acid) replication is a complex biological process that occurs in all living organisms and is responsible for copying their DNA to maintain the same set of DNA in each cell of the organism. This process starts when a cell is ready to produce two daughter cells and involves a set of proteins and other factors that are regulated in a complex way to bring out the absolute copy of an existing DNA double strand. DNA polymerase is the vital enzyme (catalytic protein) which catalyzes DNA replication. DNA polymerase uses one strand of the DNA called parent to synthesize the complementary (daughter) strand, a process referred to as semiconservative replication, by incorporating the complementary nucleotides one after the other. However, a DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis, a short oligonucleotide fragment of DNA or RNA, called a primer, must be created and paired with the template DNA strand. Directionality of DNA strand has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end of a DNA strand. DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from two of the three total phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleoside triphosphates (dNTPs). When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester bond that attaches the remaining phosphate to the growing chain. DNA replication can also be replicated in vitro (artificially, outside a cell). DNA polymerases, isolated from cells, and artificially synthesized DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA. A reaction mixture containing template DNA copies, target region specific primers, DNA polymerase, dNTPs and other co-factors is cycled between three different temperatures causing the DNA to denature (94 - 98° C) then anneal (50-65° C) and elongate/extend (72° C) using primers and a thermostable DNA polymerase like Taq Polymerase (Thermus aquaticus DNA polymerase) thereby copying (amplifying) a specific target sequence (a specific region of double stranded DNA called amplicon) to billion fold within 30-35 cycles in order to detect a discernable signal (detection) or for further use of the amplified target sequence for cloning or sequencing purposes. Another such common laboratory technique is isothermal nucleic acid amplification. This technique is also similar to PCR except instead of cycling between three different temperatures, the nucleic acid amplification is carried out at a single temperature (55-65° C) for a set period of time employing one or more enzymes that can denature and replicate the target double stranded DNA sequence. These techniques have become a cornerstone of molecular biology and is extensively used worldwide for Nucleic Acid Testing (NAT) based in vitro diagnostics, biomedical research, forensics, etc. PCR or isothermal amplification can be tailored to give quantitative or qualitative data as required depending on the analysis technique used. End-point PCR or isothermal amplification is a qualitative method wherein the nucleic acid amplification is performed and the nucleic acid amplification reacted mix is analyzed post -process using gel electrophoresis or by simple addition of a DNA intercalating dye like SYBR green I dye which turns from orange to green in presence of excessive double stranded DNA (amplicons) to check for the presence or absence of a specific target amplicon that indicates a specific condition like presence/absence of a pathogen in the sample or a mutation in the genome. Realtime or quantitative real-time PCR (qPCR) is a variant of PCR method which uses fluorescence based detection. The fluorescence increases as DNA is amplified and this in-turn will be monitored in real-time using optical measurement setup as the PCR proceeds. However, real-time PCR is expensive due to the cost of instrument and fluorescence reagents (intercalation fluorescent dyes or fluorophore labeled primers) that must be used in the reaction mixture in order to monitor the nucleic acid amplification in real-time. DNA hybridization assay based NAT is also performed for detection and quantification of a target sequence, derived directly from a DNA/RNA in a sample or indirectly using pre-amplified DNA.

By nature, the incorporation of a deoxyribonucleotide triphosphate (dNTP) into a growing DNA strand by DNA polymerase involves the formation of a covalent bond and the release of pyrophosphate and a positively charged hydrogen ion. It was previously known that protons are also a nucleic acid amplification product and that qPCR can be achieved by monitoring proton release using the changes in Current- Voltage (I-V curve) characteristics of chemical field effect transistors (solid state semiconductor sensor), preferably in a low reaction volume chamber.

OBJECT OF THE INVENTION:

It is an object of the present invention to provide an apparatus and method for monitoring nucleic acid amplification that overcomes the deficiencies of the prior arts.

Another object of the present invention is to provide an apparatus to perform NAT by end-point PCR or qPCR or isothermic amplification or DNA hybridization using solid state sensors, thermal cycling elements, control elements, control circuitry, etc.

Yet another object of this invention is to provide a method to perform the end-point PCR assay or qPCR or isothermic amplification monitoring or DNA hybridization assay using aforesaid components.

These and other objects of the invention will be apparent to those skilled in the art from the description that follows. SUMMARY OF THE INVENTION

The present invention arises due to the realization by the inventors that alternative low- cost and simpler solid state semiconductor capacitor (sensor) based apparatus can be employed to monitor nucleic acid amplification in real-time in order to quantify target nucleic acid sequence in a sample. It was further realized that the monitoring of PCR can be achieved through the change in the DNA concentration, the amount of which increases as the reaction proceeds due to amplification of DNA, which in turn affects the sensor's Capacitance- Voltage (C-V curve) characteristics. This can be used as a parameter to monitor the PCR or isothermic reaction in real-time.

According to the present invention, a system and a method for monitoring nucleic acid amplification in real-time is disclosed. The system comprises a test cartridge, a Digital to Analog Converter (DAC)biasing voltage circuit, and a controlled thermal element. The test cartridge comprises a plurality of reaction chambers, wherein each reaction chamber encloses an electrolyte insulator semiconductor capacitor (EISCAP) sensor, said reaction chamber has a provision to hold a liquid sample comprising a nucleic acid amplification reaction mixture necessary for amplification of a target nucleic acid sequence if the target nucleic acid sequence is present, said liquid sample is in contact with an ion-sensitive layer of the EISCAP sensor. The DAC biasing voltage circuit applies a bias voltage, V, across the EISCAP sensor resulting in change of capacitance, C, of EISCAP sensor, wherein a positive terminal is in contact with the liquid sample and a negative terminal is in contact with the EISCAP sensor, said change in capacitance value is read by a controller and plotted against said bias voltage for generating a C-V curve. The positive terminal is Ag/AgCl wire contacts. The liquid sample in the reaction chamber is sealed with a lid or a cap. The controlled thermal element is placed in close proximity to the reaction chamber for subjecting liquid sample to predefined temperatures for predefined time durations. The target nucleic acid sequence, if present in the liquid sample, starts to amplify and produce multiple copies of the target nucleic acid sequence upon application of the predefined temperatures resulting in increase in DNA concentration which results in shift in the C-V curve in real-time.

According to an embodiment of the present invention, a system is described, wherein the nucleic acid amplification reaction is polymerase chain reaction, wherein the liquid sample is subjected to plurality of reaction cycles and plurality of C-V curves are plotted for predefined cycles.

According to yet another embodiment of the present invention, a system is described wherein the nucleic acid amplification reaction is an isothermic reaction and wherein the plurality of C-V curves are plotted at predefined time intervals.

The liquid sample is subjected to plurality of reaction conditions and plurality of C-V curves are plotted for predefined conditions at predefined time intervals.

The plurality of reaction chambers comprises of at least one reaction chamber for holding a test liquid sample and at least one reaction chamber for holding a reference liquid sample, said test liquid sample and reference liquid sample are in contact with the ion-sensitive layer of the EISCAP sensor positioned in respective reaction chambers.

The system further comprises a controller for regulating the temperature of the controlled thermal element for predefined time intervals. In addition, the controller controls the function of the DAC biasing voltage circuit and measures the change in capacitance of EISCAP sensor with respect to the bias voltage. The controlled thermal element transfers thermal energy to the liquid sample in the reaction chamber present in the test cartridge via a plurality of thermal conduits.

According to an embodiment of the present invention, the capacitance and the bias voltage are transmitted to an external device for storage, monitoring and assessment. According to another embodiment of the present invention, a method for monitoring nucleic acid amplifications is disclosed. The method comprises the steps of preparation, loading of a liquid sample in the respective reaction chambers and initiation of the process in a reaction chamber; applying a bias voltage across an electrolyte insulator semiconductor capacitor, EISCAP sensor, the top layer which is ion-sensitive is in contact with the liquid sample, said application of bias voltage resulting in change of capacitance of EISCAP sensor; plotting of capacitance value against said bias voltage to obtain a C-V curve; wherein a target nucleic acid sequence, if present in the liquid sample, starts to amplify and produce multiple copies of the target nucleic acid sequence upon application of the predefined temperature conditions to the liquid sample for predefined time durations resulting in increase in DNA concentration which results in shift in the C-V curve in real-time.

According to an embodiment, the liquid sample is subjected to a plurality of polymerase chain reaction, PCR cycles and plurality of C-V curves are plotted for predefined temperature and time points during PCR cycles; or an isothermic reaction condition for a predefined time period and plurality of C-V curves are plotted at predefined time points during this time period.

BRIEF DESCRIPTION OF THE DRAWINGS

A clear understanding of the key features of the invention summarized above may be had by reference to the appended drawings, which illustrate the method and system of the invention, although it will be understood that such drawings depict preferred embodiments of the invention and, therefore, are not to be considered as limiting its scope with regard to other embodiments which the invention is capable of contemplating.

Figure 1A exemplifies the basic configuration of an EISCAP sensor, according to an embodiment of the present invention. Figure 1B-1C show cross-sectional views of the test cartridge according to an embodiment of the present invention.

Figure ID is a top view of the top unit and bottom unit casings of the test cartridge, according to yet another embodiment of the present invention.

Figure 2 is a block diagram of the system, according to an embodiment of the present invention.

Figure 3A shows the three modes by which amplification process can be initiated, according to an embodiment of the present invention.

Figure 3B shows the steps carried out in an amplification process, according to an embodiment of the present invention.

Figure 4 represents schematic of the system, according to an embodiment of the present invention.

Figure 5A-5G shows various stages of Capacitance-Voltage curves during the process of amplification, according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, there is shown an illustrative embodiment of the system and method as disclosed in the disclosure. It should be understood that the disclosure is susceptible to various modifications and alternative forms; specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It will be appreciated as the description proceeds that the disclosure may be used in other types of appliances and may be realized in different embodiments.

Before describing embodiments in detail, it may be observed that the novelty and inventive step that are in accordance with the present disclosure reside in the system and method as disclosed, accordingly, the drawings are showing only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein. The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a setup, device or system that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such setup, device or system. In other words, one or more elements in a system or apparatus proceeded by "comprises... a" does not, without more constraints, preclude the existence of other elements or additional elements in the system or apparatus. The following paragraphs explain various aspect of the present disclosure with reference to figures.

The present invention is based on the specific sensing capabilities of an Electrolyte- Insulator-Semiconductor Capacitor (EISCAP), a solid state sensor. A basic EISCAP sensor device is depicted in Figure 1A showing various components of the device. EISCAP sensor consists of a semiconductor with a native oxide interface 105 and ion sensitive layer/insulator 110. The ion sensitive layer 110 is sensitive to charged species such as hydrogen ions (protons) or DNA (negative charge) present or generated in the electrolyte or the liquid sample. The change in DNA concentration can be measured as the shift in flat-band voltage in the linear region of the Capacitance-Voltage curve (C-V curve). As the DNA concentration in the analyte increases, the flat-band voltage becomes increasingly positive (right-sided shift) as illustrated in Figure 5B. The shift in flat -band voltage (V) as measured before and after a nucleic acid amplification reaction can give a qualitative end-point result. This is illustrated in Figures 5E and 5F. Similarly, a continuous assessment of changes in flat-band voltage during nucleic acid amplification corresponding to accumulation of net DNA negative charge as the reaction proceeds can be carried out using the EISCAP sensor for real-time measurement.

According to the present invention, a test cartridge is provided with a plurality of reaction chambers. Each reaction chamber encloses an electrolyte insulator semiconductor capacitor (EISCAP). The reaction chamber has a provision to hold a liquid sample which comprises of a nucleic acid amplification reaction mixture necessary for amplification of a target nucleic acid sequence. The liquid sample is in contact with the ion-sensitive layer 110 of the EISCAP sensor.

Figure IB shows a cross-sectional view of the test cartridge 100, according to an embodiment of the present invention. For the ease of understanding, the test cartridge illustrated here has only one reaction chamber comprising of one EISCAP sensor and provision to hold one liquid sample. The test cartridge comprises of plurality of such reaction chambers.

As shown in the figure IB, an EISCAP sensor 115 (bare die) is encased between a top unit casing 120 and a bottom unit casing 125 of the test cartridge 100. In another embodiment, the test cartridge 100 is a single mould with an EISCAP sensor 115 enclosed therein. The top unit casing 120 is provided with a reaction chamber 130 which holds a liquid sample. The placement of liquid sample is such that the liquid sample is in contact with the ion-sensitive layer 110 of the EISCAP sensor 115. The reaction chamber 130 is sealed by a lid/cap 135 after a liquid sample is dispensed into it.

The test cartridge 100 is provided with a positive contact port 140 and a negative contact port 145. The positive contact port 140 connects positive electrode to the liquid sample present in the reaction chamber 130. The positive contact port 140 is moulded in such a way that a channel connects the inner wall of the reaction chamber 130. A negative electrode contacts the EISCAP sensor 115 via a negative contact port 145. In a preferred embodiment, the positive electrode is Ag/AgCl and is sealed by either thermal sealing or chemical sealing. Thermal sealing is done by pinching the outer wall of the reaction chamber 130 with a hot metal at 120°C or greater with the metal in periphery of the channel. Chemical sealing is done by utilizing chemical waterproof sealants such as polydimethylsiloxane polymer or silicone polymer or any elastomer with waterproof property. Figure IB, illustrates a height filler material 150 used below the EISCAP sensor 115 to fill a gap in the mould. A through hole is made through this height filler material 150 such that the negative electrode can contact the EISCAP sensor 115 without hindrance. The height filler material 150 can be elastomer such as silicone, plastic such as polyethylene, inert glass, etc.

Figure 1C shows a cross-sectional view of the test-cartridge 100, according to an embodiment of the present invention. In this test-cartridge 100, an EISCAP sensor 115a bonded with a glass coated with Ag/AgCl electrode paste or ink replaces the EISCAP sensor 115 and the height filler material 150 of Figure IB. This EISCAP sensor 115a is bonded to one surface of the glass with Ag/AgCl electrode paste or ink coated on the other surface of the glass and is encased between the top unit casing 120 and the bottom unit casings 125 of the test cartridge 100.

Figure ID shows a top view of the top unit casings 1200 and bottom unit casings 1250 of the test cartridge 1000 in accordance with an embodiment of the present invention. As seen in the Figure, the overall structure of the test cartridge 1000 is flat and coin like shaped. The test cartridge 1000 can be assembled and directly placed in a fitting thermal conduit. The larger flat surface provides a larger heat transfer area. There is a negative contact port 1450 and a positive contact port 1400 which are channels that run from the exterior all the way to the interior of the test cartridge 1000. The negative contact runs along negative contact port 1450 and contacts the EISCAP sensor 1150. The positive contact runs along the positive contact port 1400 and contacts the reaction chamber 1300.

Figure 2 illustrates an exemplary embodiment of nucleic acid amplification testing equipment (EISLAB Control Unit) with a test cartridge. A controlled thermal element 210 generates thermal energy and regulates the thermal nature of nucleic acid amplification process. Plurality of thermal conduits 215 transfer the generated thermal energy from the controlled thermal element 210 to the test cartridge 205. The plurality of thermal conduits 215 are made of good thermal conductive material which may be aluminium, copper, gold, silver, tungsten and their alloys thereof. One or more heat sinks 225 is attached to the controlled thermal element 210 which may or may not have one or more heat sink coupled fan to vent out heat or maintain required thermal conditions and one or more heat sink frame 220 is provided for excess thermal dissipation and secure placement of test cartridge 205. The one or more heat sinks 225 are made of good thermal conductor material which may be aluminium, copper, gold, silver, tungsten, and their alloys thereof and is insulated electrically from other parts of the EISLAB Control unit 200. It prevents heat build-up within the EISLAB Control Unit 200 and provides efficient overall working. A power supply unit 230 powers up; the electric components in the EISLAB Control unit 200. Similarly, a battery can also be incorporated as an alternative to the direct AC supply which makes the EISLAB Control unit 200 portable. There is a communication unit 235 linked with the EISLAB Control unit 200 to output data such as to a compatible portable electronic component, mobile phone, electronic tablet or the like. Data generated by the EISLAB Control unit 200 is also stored within the system or an external storage device or a cloud.

Referring to Figure 3A, there are three modes by which amplification process of the liquid sample can be initiated. For example, in a first mode, an automated module can be integrated with the present Nucleic acid amplification testing equipment (EISLAB Control Unit). In this equipment, sample processing is done in an automated manner and the pure nucleic acid extract is mixed with the nucleic acid amplification reaction mixture and then delivered to the reaction chamber automatically. Alternatively, the pure nucleic acid extract obtained through automated sample processing can be delivered into the reaction chamber containing pre-lyophilized nucleic acid amplification component mixture in an automatically or manually. In a second mode, the liquid sample can be manually processed outside the aforementioned equipment using available nucleic acid extraction kits or by any other means and the final isolated pure DNA/RNA can be mixed with nucleic acid amplification mixture and then placed in the reaction chamber, or added into the reaction chamber containing pre-lyophilized nucleic acid amplification mixture In a third mode, crude(unprocessed) sample can be mixed manually with nucleic acid amplification mixture and then placed in the reaction chamber or the crude sample can be dispensed directly into the reaction chamber containing pre-lyophilized nucleic acid amplification mixture. Here the pre-lyophilized nucleic acid amplification mixture containing reaction chamber can be prepared in two ways: 1) nucleic acid amplification mixture lyophilized first (such as a small tablet or bead) and then loaded in the reaction chamber; or 2) the reaction chamber with the nucleic acid amplification mixture is lyophilized together (such as a thin smear wall on the interiors of the reaction chamber). When the liquid sample is placed, the lyophilized nucleic acid amplification mixture is rehydrated and become amenable to perform nucleic acid amplification reaction.

Referring to Figure 3B, the liquid sample is placed in a reaction chamber which is sealed by a lid. The test cartridge is placed over a heat sink frame 220 and secured in position. At step 305, the system is turned on and parameters for nucleic acid amplification set. The parameters may be, but not limited to, liquid sample volume, thermal conditions, time duration, etc. At step 310, the system is initiated upon applying thermal conditions by controlled thermal element to the liquid sample. As shown in step 325, at particular intervals, capacitance-voltage data is collected and analysed by a controller for the bias voltage data point at 70% normalized capacitance by the system. At step 330, the secondary data is plotted which can be visualized in the communication display.

Alternatively, the raw data which is the capacitance-voltage data is stored in a local storage device or a remote storage which can be over a cloud. The secondary data is plotted after analysis from back-end processing unit such as cloud computing or remote computing device. This data is displayed on communication module. This reduces the processing load of the controller. The communication module can be a mobile phone, tablet, computer, display device or the like.

At step 315, after the set processing time or cycle, nucleic acid amplification process ends. At step 320, the test cartridge is discarded.

Referring to Figure 4, EISLAB Control Unit 400 performs two functions - one function is to perform thermal cycling or maintain isothermal conditions and the second function is the capacitance measurement. A controlled thermal element 405 is used as a heating element and to perform the thermal cycling. Power is provided to the controlled thermal element 405 by a power supply unit 430. The controlled thermal element 405 works by consuming power and heats or cools based on the polarity of current supplied to it.

A temperature sensor 410 is attached to the controlled thermal element405 which tracks change in temperature. This change in temperature is converted to a corresponding analog voltage. This analog voltage is fed to a controller 420 via an analog to digital converter (ADC) 415. The controller 420 is programmed in such a way that this analog voltage is back calculated to temperature of the controlled thermal element 405. This temperature is the actual/current value and this value is compared with value set by the user. This value can be set before the thermal cycling process starts. Depending on the difference in temperature, the controller 420 generates Pulse Wave Modulation (PWM) signal for cooling or heating. The PWM signal from the controller 420 is given to a MOSFET driver 425. This MOSFET driver 425 drives the controlled thermal element 405. The MOSFET driver 425 can also reverse the polarity of current to the controlled thermal element 405. The controlled thermal element 405 uses a Proportional Integral Derivative (PID) algorithm which is programmed into it. The temperature gain values set for the controller 420 determine how fast the controller420 can track the change in temperature and bring it to the set value accurately. These temperature gain values are found out by trial and error.

As an example of an isothermal amplification reaction, at first the EISLAB Control Unit 400 is switched ON and user sets the temperature value to 64°C.for a predetermined time period of 60 minutes. When the EISLAB Control Unit 400 starts running and the temperature sensor 410 reads the temperature at the controlled thermal element 405. This temperature is fed back to the controller 420. The temperature value at this instant is an ambient temperature as the machine has just started to run. This temperature value is compared with the value set by the user. Based on the difference, the controlled thermal element 405 transmits PWM signals which drives the MOSFET driver 425 to conduct in one direction and provide signal to the controlled thermal element 405 to generate heat. The values set by user, while tuning on the EISLAB Control Unit 400, determines the speed of tracking of performance of the EISLAB Control Unit 400. Once the value set by the user is reached, the MOSFET driver gives sufficient current to controlled thermal element to maintain a temperature value at 64 °C for 60 minutes.

The second function of the EISLAB Control Unit 400 is the measurement of capacitance. In this regard, an example of isothermal amplification reaction at 64°C is considered. The nucleic acid amplification reaction mixture containing a DNA sample (or known DNA as positive control) is placed in the reaction chamber on the EISCAP sensor 445 and exposed to a temperature of 64°C.C At this temperature, the DNA amplification process is initiated. At this stage, for the EISCAP sensor 445 to function, it must be biased. The bias voltage is taken from the controller 420 via a biasing voltage circuit which is a digital to analog converter (DAC) 440. The biasing voltage circuit 440 may be a 12 bit one or the like, and every bit change of the biasing voltage circuit 440 corresponds to 20 millivolt change in analog. Thus, the EISCAP sensor445 is biased from -3V to 4V. The voltage is increased in steps of 20mV. When biasing starts, EISCAP sensor 445 starts to change its capacitance. This change in capacitance may be detected by a customized IC 'C-V converter'which converts it into voltage. This voltage is given to the controller 420 via an inbuilt analog to digital converter. The controller 420 now back calculates the capacitance value. Once the capacitance values are found out, it is plotted against the bias voltage to get a C-V curve. Fig,5A illustrates a C-V curve for a characterized liquid sample. Varying the voltage in a particular range provides capacitance variation which in turn gives the data on the constituents of the charge carriers in liquid sample held in the reaction chamber. The capacitance measurements are in nano-Farad to pico-Farad range. For analysis, the raw data is normalized. That is the capacitance for every C-V plot is normalized (Every data point divided by the maximum of capacitance value (data point) obtained) and the corresponding voltage values at capacitance 70% is read (linear portion of the C-V plot is between 40% to 70%).

Figure 5B illustrates C-V curves depicting C-V response for advancing nucleic acid amplification during PCR. Increasing DNA content due to amplification increases net negative charge of the reaction mixture thereby shifting the C-V curve to the right-hand side. The same principle is utilized to monitor isothermal nucleic acid amplification reactions also where the C-V curve shifts to the right-hand side with reaction time as in during a Loop Mediated amplification (LAMP).

In case of thermal cycling, the capacitance measurements are obtained in every cycle during the 'annealing' step. Figure5C illustrates a primary plot of C-V for end point measurements at every 10^th cycle of a 40 cycle PCR run (typical experiment result obtained from an end-point PCR experiment where aliquots of a PCR reaction mix where subjected to different number of PCR cycles (10 cycle intervals). The region of interest is 70% of the normalized capacitance. The corresponding bias voltage, V, are noted for each C-V curve in the C-V plot. A secondary plot is derived from the primary plot as shown in Figure 5D, where 5D differential voltage, AV (bias voltage at time of measurement (V at T_m) - reference bias voltage at initial conditions (V at To) is plotted against PCR cycle number.

Figures 5E and 5F exemplify the C-V responses for increasing known concentrations of DNA (both single stranded and double stranded) obtained by end-point measurements and thereby is a proof of the principle used in this invention. In Figure 5E, a primary plot showing 70% of the normalized capacitance and the corresponding bias voltage in C-V curve for different known concentrations (standards) of single stranded DNA (ssDNA) as well as a secondary plot showing differential voltage, AV (bias voltage reading at each ssDNA concentration - reference-r bias voltage for buffer solution without DNA) is plotted for different concentrations of ssDNA. ssDNA is 22 nucleotide in length. ssDNA concentration is ranging from 0 - 25 micromolar.

Figure 5F, illustrates a graph for differential voltage (bias voltage V reading at each DNA concentration - reference-r bias voltage for buffer solution without DNA) for different known concentrations (standards) of double stranded DNA (dsDNA) ranging from 1 femtogram per microliter to 1 microgram per microliter.

Figure 5G, illustrates a graph which shows real-time data obtained for LAMP, isothermal amplification based tuberculosis (TB) test done with nucleic acid amplification test cartridge and equipment. Differential voltage, AV (bias voltage reading at time of measurement (V at T_m) - reference-r bias voltage reading at initial conditions time (V at To). Clear difference is observed between Negative and Positive control by 50-100 mV from 25minutes onwards.

These final plots are displayed in a display unit 465, 475 as shown in Figure 5. The display unit 465, 475 may be a portable electronic display unit or a portable electronic device such as tablet, mobile phone or the like. The display unit 465, 475 is connected to the controller 420 in two ways - one in a computer via Universal asynchronous receiver transmitter (UART) 460 and the other way is to communicate it to the portable electronic device via a bridge IC 470. This IC 470 acts as a bridge between the embedded device connecting the controller 420 and the display unit 475. The final plot is displayed at the respective electronic display or portable electronic device in realtime. The electronic display or portable electronic device is also used by the user to set various values such as denaturation/extension/annealing temperatures and respective holding time for each step in case of polymerase chain reaction (e.g., 35 cycles of 95 °C for 20 sec, 64°C for 20 sec and 72°C for 20 sec). In case of Isothermal amplification, the isothermic reaction temperature and holding time (e.g., hold 64°C for 60 min). The user may alternatively select pre-programmed conditions according to the test performed, e.g., Malaria detection program or TB detection program. To make it easier, an application programmed in the portable electronic device may help the user by automatically loading the specific program from the memory of the portable electronic device.

While the particular preferred embodiments of the present disclosure have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the teachings of the disclosure. It is therefore contemplates that the present disclosure cover any and all modifications, variations or equivalents that fall within the scope of the basic underlying principles disclosed above and claimed herein.

While the present invention has been described in terms of particular embodiments and applications, in both summarized and detailed forms, it is not intended that these descriptions in any way limit its scope to any such embodiments and applications, and it will be understood that many substitutions, changes and variations in the described embodiments, applications and details of the method and system illustrated herein and of their operation can be made by those skilled in the art without departing from the spirit of this invention.

References:

1. Hareesh Vemulachedu et al (2009), J. Mater. Sci: Mater. Med; 20: S229-S234

2. Tsung Wu Lin et al (2010), Biosensors and Bioelectronics; 25: 2706-2710.

3. Toumazou et al. US 788015 (2011)

4. Mohanasundaram SV et al. (2013) IEEE Sensor Journal; 13(5) 1941-1948

Claims

We Claim:

1. A system for monitoring nucleic acid amplification in real-time, the system comprising:

a test cartridge comprising a plurality of reaction chambers, wherein each reaction chamber encloses an electrolyte insulator semiconductor capacitor (EISCAP), said reaction chamber having a provision to hold a liquid sample comprising a nucleic acid amplification reaction mixture necessary for amplification of a target nucleic acid sequence if the target nucleic acid sequence is present, said liquid sample is in contact with an ion-sensitive layer of the EISCAP sensor;

a DAC biasing voltage circuit applying a bias voltage, V, across the EISCAP sensor resulting in change of capacitance, C, of EISCAP, wherein a positive terminal is in contact with the liquid sample and a negative terminal is in contact with the EISCAP, said change in capacitance value is read by a controller and plotted against said bias voltage for generating a C-V curve; and

a controlled thermal element placed in close proximity to the reaction chamber for subjecting liquid sample to predefined temperatures for predefined time durations;

wherein the target nucleic acid sequence, if present in the liquid sample, starts to amplify and produce multiple copies of the target nucleic acid sequence upon application of the predefined temperatures resulting in increase in DNA concentration which results in shift in the C-V curve in real-time.

2. The system of claim 1, wherein the nucleic acid amplification reaction is Polymerase chain reaction.

3. The system of claim 1, wherein the nucleic acid amplification reaction is isothermic reaction.

4. The system of claim 2, wherein the liquid sample is subjected to plurality of reaction cycles and plurality of C-V curves are plotted for predefined cycles.

5. The system of claim 3, wherein plurality of C-V curves are plotted at predefined time intervals.

6. The system of claim 1, wherein the plurality of reaction chambers comprises of at least one reaction chamber for holding test sample and at least one reaction chamber for holding a reference sample, said test sample and reference sample is in contact with the ion-sensitive layer of the EISCAP sensor positioned in respective reaction chambers.

7. The system of claim 1, wherein the positive terminal is Ag/AgCl wire contacts.

8. The system of claim 1, wherein the controller regulates the temperature of the controlled thermal element for predefined time intervals.

9. The system of claim 1, wherein the controlled thermal element transfers thermal energy to the liquid sample in the reaction chamber present in the test cartridge via a plurality of thermal conduits.

10. The system of claim 1, wherein the liquid sample in the reaction chamber is sealed with a lid or a cap.

11. The system of claim 1 , further comprising of heat sinks for maintaining or lowering of temperature of the controlled thermal element.

12. The system of claim 1, wherein the controller controls the function of the DAC biasing voltage circuit and measures the change in capacitance of EISCAP with respect to the bias voltage.

13. The system of claim 1, wherein the capacitance and the bias voltage are transmitted to external devices for storage, monitoring and assessment.

14. A method for monitoring nucleic acid amplification comprising the steps of: preparation, loading of a liquid sample into the respective reaction chambers and initiation of the process in a reaction chamber;

applying a bias voltage across an electrolyte insulator semiconductor capacitor, EISCAP sensor, sensing layer of which is in contact with the liquid sample, said application of bias voltage resulting in change of capacitance of EISCAP;

plotting of capacitance value against said bias voltage to obtain a C-V curve; and

wherein the target nucleic acid sequence, if present in the liquid sample, starts to amplify and produce multiple copies of the target nucleic acid sequence upon application of predefined temperatures to the liquid sample for predefined time durations resulting in increase in DNA concentration which results in shift in the C-V curve in real-time.

15. The method of claim 14, wherein said liquid sample is subjected to:

a) plurality of polymerase chain reaction (PCR) cycles and plurality of C-V curves are plotted at predefined temperature and time points during PCR cycles; or

b) isothermic reaction condition for a predefined time period and plurality of C- V curves are plotted at predefined time points during this time period.