An integrated circuit for chip-based analysis of enzyme kinetics and metabolite quantification

We have created a novel chip-based diagnostic tools based upon quantification of metabolites using enzymes specific for their chemical conversion. Using this device we show for the first time that a solid-state circuit can be used to measure enzyme kinetics and calculate the Michaelis-Menten constant. Substrate concentration dependency of enzyme reaction rates is central to this aim. Ion-sensitive field effect transistors (ISFET) are excellent transducers for biosensing applications that are reliant upon enzyme assays, especially since they can be fabricated using mainstream microelectronics technology to ensure low unit cost, mass-manufacture, scaling to make many sensors and straightforward miniaturisation for use in point-of-care devices. Here, we describe an integrated ISFET array comprising 216 sensors. The device was fabricated with a complementary metal oxide semiconductor (CMOS) process. Unlike traditional CMOS ISFET sensors that use the Si3N4 passivation of the foundry for ion detection, the device reported here was processed with a layer of Ta2O5 that increased the detection sensitivity to 45 mV/pH unit at the sensor readout. The drift was reduced to 0.8 mV/hour with a linear pH response between pH 2 – 12. A high-speed instrumentation system capable of acquiring nearly 500 fps was developed to stream out the data. The device was then used to measure glucose concentration through the activity of hexokinase in the range of 0.05 mM – 231 mM, encompassing glucose’s physiological range in blood. Localised and temporal enzyme kinetics of hexokinase was studied in detail. These results present a roadmap towards a viable personal metabolome machine

Metabolomics on integrated circuit

We have demonstrated a chip-based diagnostics tool for the quantification of metabolites, using specific enzymes, to study enzyme kinetics and calculate the Michaelis-Menten constant. An array of 256×256 ion-sensitive field effect transistors (ISFETs) fabricated in a complementary metal oxide semiconductor (CMOS) process is used for this prototype. We have used hexokinase enzyme reaction on the ISFET CMOS chip with glucose concentration in the physiological range of 0.05 mM – 231 mM and successfully studied the enzyme kinetics of hexokinase in detail. This will promote future research towards multiplexing enzyme-based metabolite quantification on a single chip, ultimately opening a pathway towards a personal metabolome machine

Hybrid dual mode sensor for simultaneous detection of two serum metabolites

Metabolites are the ultimate readout of disease phenotype that plays a significant role in the study of human disease. Multiple metabolites sometimes serve as biomarkers for a single metabolic disease. Therefore, simultaneous detection and analysis of those metabolites facilitate early diagnostics of the disease. Conventional approaches to detect and quantify metabolites include mass spectrometry and nuclear magnetic resonance that require bulky and expensive equipment. Here, we present a disposable sensing platform that is based on complementary metal–oxide–semiconductor process. It contains two sensors: an ion sensitive field-effect transistor and photodiode that can work independently for detection of pH and color change produced during the metabolite-enzyme reaction. Serum glucose and cholesterol have been detected and quantified simultaneously with the new platform, which shows good sensitivity within the physiological range. Low cost and easy manipulation make our device a prime candidate for personal metabolome sensing diagnostics

A colorimetric CMOS-based platform for rapid total serum cholesterol quantification

Elevated cholesterol levels are associated with a greater risk of developing cardiovascular disease and other illnesses, making it a prime candidate for detection on a disposable biosensor for rapid point of care diagnostics. One of the methods to quantify cholesterol levels in human blood serum uses an optically mediated enzyme assay and a bench top spectrophotometer. The bulkiness and power hungry nature of the equipment limits its usage to laboratories. Here, we present a new disposable sensing platform that is based on a complementary metal oxide semiconductor process for total cholesterol quantification in pure blood serum. The platform that we implemented comprises readily mass-manufacturable components that exploit colorimetric changes of cholesterol oxidase and cholesterol esterase reactions. We have shown that our quantification results are comparable to that obtained by a bench top spectrophotometer. Using the implemented device, we have measured cholesterol concentration in human blood serum as low as 29 μM with a limit of detection at 13 μM, which is approximately 400 times lower than average physiological range, implying that our device also has the potential to be used for applications that require greater sensitivity

An integrated portable system for single chip simultaneous measurement of multiple disease associated metabolites

Metabolites, the small molecules that underpin life, can act as indicators of the physiological state of the body when their abundance varies, offering routes to diagnosis of many diseases. The ability to assay for multiple metabolites simultaneously will underpin a new generation of precision diagnostic tools. Here, we report the development of a handheld device based on complementary metal oxide semiconductor (CMOS) technology with multiple isolated micro-well reaction zones and integrated optical sensing allowing simultaneous enzyme-based assays of multiple metabolites (choline, xanthine, sarcosine and cholesterol) associated with multiple diseases. These metabolites were measured in clinically relevant concentration range with minimum concentrations measured: 25 μM for choline, 100 μM for xanthine, 1.25 μM for sarcosine and 50 μM for cholesterol. Linking the device to an Android-based user interface allows for quantification of metabolites in serum and urine within 2 min of applying samples to the device. The quantitative performance of the device was validated by comparison to accredited tests for cholesterol and glucose

Hybrid Amperometric and Potentiometric Sensing Based on a CMOS ISFET Array

Potentiometry and amperometry are some of the most important techniques for electroanalytical applications. Integrating these two techniques on a single chip using CMOS technology paves the way for more analysis and measurement of chemical solutions. In this paper, we describe the integration of electrode transducers (amperometry) on an ion imager based on an ISFET array (potentiometry). In particular, this integration enables the spatial representation of the potential distribution of active electrodes in a chemical solution under investigation

The Multicorder: A Handheld Multimodal Metabolomics-on-CMOS Sensing Platform

The use of CMOS platforms in medical point-of-care applications, by integrating all steps from sample to data output, has the potential to reduce the diagnostic cost and the time from days to seconds. Here we present the `Multicorder' technology, a handheld versatile multimodal platform for rapid metabolites quantification. The current platform is composed of a cartridge, a reader and a graphic user interface. The sensing core of the cartridge is the CMOS chip which integrates a 16×16 array of multi-sensor elements. Each element is composed of two optical and one chemical sensor. The platform is therefore capable of performing multi-mode measurements: namely colorimetric, chemiluminescence, pH sensing and surface plasmon resonance. In addition to the reader that is employed for addressing, data digitization and transmission, a tablet computer performs data collection, visualization, analysis and storage. In this paper, we demonstrate colorimetric, chemiluminescence and pH sensing on the same platform by on-chip quantification of different metabolites in their physiological range. The platform we have developed has the potential to lead the way to a new generation of commercial devices in the footsteps of the current commercial glucometers for quick multi-metabolite quantification for both acute and chronic medicines

Multimodal integrated sensor platform for rapid biomarker detection

Precision metabolomics and quantification for cost-effective, rapid diagnosis of disease are key goals in personalized medicine and point-of-care testing. Presently, patients are subjected to multiple test procedures requiring large laboratory equipment. Microelectronics has already made modern computing and communications possible by integration of complex functions within a single chip. As More than Moore technology increases in importance, integrated circuits for densely patterned sensor chips have grown in significance. Here, we present a versatile single CMOS chip forming a platform to address personalized needs through on-chip multimodal optical and electrochemical detection that will reduce the number of tests that patients must take. The chip integrates interleaved sensing subsystems for quadruple-mode colorimetric, chemiluminescent, surface plasmon resonance and hydrogen ion measurements. These subsystems include a photodiode array and a single photon avalanche diode array, with some elements functionalized to introduce a surface plasmon resonance mode. The chip also includes an array of ion sensitive field effect transistors. The sensor arrays are distributed uniformly over an active area on the chip surface in a scalable and modular design. Bio-functionalization of the physical sensors yields a highly selective simultaneous multiple-assay platform in a disposable format. We demonstrate its versatile capabilities through quantified bioassays performed on-chip for glucose, cholesterol, urea and urate, each within their naturally occurring physiological range

Metabolomic sensing system for personalised medicine using an integrated CMOS sensor array technology

Precision healthcare, also known as personalised medicine, is based on our understanding of the fundamental building blocks of biological systems, with the ultimate aim to clinically identify the best therapeutic strategy for each individual. Genomics and sequencing technologies have brought this to the foreground by enabling an individual’s entire genome to be mapped for less than a thousand dollar in just one day. Recently, metabolomics, the quantitative measurement of small molecules, has emerged as a field to understand an individual’s molecular profile in terms of both genetics and environmental factors. This is crucial because a genome could only indicate an individual’s susceptibility to a particular disease, whereas a metabolome provides an immediate measurement of body function, enabling a means of diagnosis. However, the current approach of measurements depends on large-scale and expensive equipment such as mass spectroscopy and NMR instrumentation, which does not offer a single analytical platform to detect the entire metabolome. This thesis describes the development of an integrated CMOS sensor array technology as a single platform to quantify different metabolites using specific enzymes. The key stages in the work were: to construct instrumentation systems to perform enzyme assays on the CMOS sensor array; to establish techniques to package the CMOS sensor array for an aqueous environment; to implement and develop a room temperature Ta2O5 sputtering process on CMOS sensor array for hydrogen ion detection; to collaborate with a chemist and investigate an inorganic layer on top of the CMOS ISFET sensor to show an improvement of sensitivity towards potassium ion; to test several different enzyme assays electrochemically and optically and show the functionalities of the sensors; to devise microfluidic channels for segregation of the sensor array into different compartments and perform enzyme immobilisation techniques on CMOS chips; and integrate the packaged chip with microfluidic channels and enzyme immobilisation using 2D inkjet printer into a complete system that has the potential to be used as a multi-enzyme platform for detection of different metabolites. Two CMOS sensor array chips (1) a 256×256-pixel ISFET array chip and (2) a 16×16-pixel Multi-Corder chip were fully understood. Therefore, a high-speed instrumentation system was constructed for the ISFET array chip with a maximum readout speed of 500 frames per second, with 2D and 3D imaging capability, as well as single pixel analysis. Follow by that, a miniaturised measurement platform was implemented for the Multi-Corder chip that has three different sensor arrays, which are ISFET, PD and SPAD. All the sensor arrays can be operated independently or together (ionic sensor and one of the optical sensors). Several post-processing steps were investigated to allow suitable fabrication process on small 4×4 mm2 CMOS chips. Post-processing of the CMOS chips was first established using room temperature sputtering process for Ta2O5 layer, achieving Ta:O ratio of 1:1.77 and a surface roughness of 0.42 nm. This Ta2O5 layer was then fabricated on top of CMOS ISFETs, which improves the ISFET pH sensitivity to 45 mV/pH, with an average drift of 6.5 ± 8.6 mV/hour from chip to chip and a working pH range of 2 to 12. Furthermore, a layer of POMs was drop casted on top of Ta2O5 ISFET to make ISFET sensitive to potassium ions. This was investigated in terms of potassium ions sensitivity, hydrogen ions sensitivity and sodium ions as interfering background ions. The POMs Ta2O5 ISFET was found to have a net potassium sensitivity of 75 mV/pK, with a linear range between pH 1.5 to 3. Moreover, the POMs ISFET has -5 mV/pH in pH sensitivity, showing that it is selectivity towards potassium ions and not hydrogen ions. However, sodium ions were found to produce a large interference towards the pK sensitivity of POMs ISFET and reduced the pK sensitivity of POMs ISFET. Hence, further work is still required to modify POMs layer for better selectivity and sensitivity. Besides that, microfluidic channels were fabricated on top of the CMOS chips that could provide segregation for multiple enzyme assays on a single chip. In addition, a PDMS and a manual dam and fill method were developed to encapsulate the CMOS chips for wet biochemistry measurements. The CMOS sensor array was found to have the ensemble averaging capability to reduce noise as a function of √N , where N is the number of sensors used for averaging. Several enzyme assays that include: hexokinase, lactate dehydrogenase, urease and lipase were tested on the ISFET sensor array. Moreover, using an optical sensor array, namely a PD on the Multi-Corder chip and using LED illumination, quantification of cholesterol levels in human blood serum was demonstrated. Enzyme kinetics calculations were performed for hexokinase and cholesterol oxidase assays and the results were comparable to that obtained from a bench top spectrophotometer. This shows the CMOS sensor array can be used as a low cost portable diagnostic device. Several enzyme immobilisation techniques were explored but were unsuccessful. Alginate enzyme gel immobilisation with a 2D inkjet printer was found to be the best candidate to bio-functionalise the CMOS sensor array. The packaged chip was integrated with microfluidic channels and alginate enzyme gel immobilisation into a complete system, in order to perform an enzyme assay with its control experiments simultaneously on a single chip. As a proof-of-concept, this complete system has the potential to be used as a multiple metabolite quantification platform