53 results found
Georgiou P, Moniri A, Moser N, et al., 2019, Devices and method for detecting an amplification event, WO2019234451A1
A method is disclosed herein for detecting an amplification reaction in a solution containing a biological sample using an array of ion sensors. The amplification reaction is indicative of the presence of a nucleic acid. The method comprises 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, and 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. The method further comprises determining, based on the comparing, that an amplification event has occurred in the solution in the vicinity of the first sensor.
Georgiou P, Malpartida Cardenas K, Yu L-S, et al., 2019, Method for detecting a single nucleotide polymorphism (snp) using lamp and blocking primers, WO2019234251A1
The present application relates to methods for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence under isothermal conditions using primers specific for said first allele, in particular using Loop mediated isothermal amplification (LAMP), wherein the amplification of a second allele is prevented by using blocking primers.
Georgiou P, Yu L-S, Malpartida-Cardenas K, et al., 2019, Method for detecting a tandem repeat, WO2019234252A1
The present application relates to methods for detecting a tandem repeat in a nucleic acid sequence under isothermal conditions using primers.
Georgiou P, Moniri A, Rodriguez Manzano J, 2019, A method for analysis of real-time amplification data, WO2019234247A1
This disclosure relates to methods, systems, computer programs and computer- readable media for the multidimensional analysis of real-time amplification data. A framework is presented that shows that the benefits of standard curves extend beyond absolute quantification when observed in a multidimensional environment. Relating to the field of Machine Learning, the disclosed method combines multiple extracted features (e.g. linear features) in order to analyse real-time amplification data using a multidimensional view. The method involves two new concepts: the multidimensional standard curve and its 'home', the feature space. Together they expand the capabilities of standard curves, allowing for simultaneous absolute quantification, outlier detection and providing insights into amplification kinetics. The new methodology thus enables enhanced quantification of nucleic acids, single-channel multiplexing, outlier detection, characteristic patterns in the multidimensional space related to amplification kinetics and increased robustness for sample identification and quantification.
Malpartida-Cardenas K, Miscourides N, Rodriguez-Manzano J, et al., 2019, Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform, Biosensors and Bioelectronics, Vol: 145, ISSN: 0956-5663
Early and accurate diagnosis of malaria and drug-resistance is essential to effective disease management. Available rapid malaria diagnostic tests present limitations in analytical sensitivity, drug-resistance testing and/or quantification. Conversely, diagnostic methods based on nucleic acid amplification stepped forwards owing to their high sensitivity, specificity and robustness. Nevertheless, these methods commonly rely on optical measurements and complex instrumentation which limit their applicability in resource-poor, point-of-care settings. This paper reports the specific, quantitative and fully-electronic detection of Plasmodium falciparum, the predominant malaria-causing parasite worldwide, using a Lab-on-Chip platform developed in-house. Furthermore, we demonstrate on-chip detection of C580Y, the most prevalent single-nucleotide polymorphism associated to artemisinin-resistant malaria. Real-time non-optical DNA sensing is facilitated using Ion-Sensitive Field-Effect Transistors, fabricated in unmodified complementary metal-oxide-semiconductor (CMOS) technology, coupled with loop-mediated isothermal amplification. This work holds significant potential for the development of a fully portable and quantitative malaria diagnostic that can be used as a rapid point-of-care test.
Terracina D, Moniri A, Rodriguez-Manzano J, et al., 2019, Real-Time forecasting and classification of trunk muscle fatigue using surface electromyography
© 2019 IEEE. Low Back Pain (LBP) affects the vast majority of the population at some point in their lives. People with LBP show altered trunk muscle activity and enhanced fatigability of trunk muscles is associated with the development and future risk of LBP. Therefore, a system that can forecast trunk muscle activity and detect fatigue can help subjects, practitioners and physiotherapists in the diagnosis, monitoring and recovery of LBP. In this paper, we present a novel approach in order to determine whether subjects are fatigued, or transitioning to fatigue, 25 seconds ahead of time using surface Electromyography (sEMG) from 14 trunk muscles. This is achieved using a three-step approach: A) extracting features related to fatigue from sEMG, B) forecasting the features using a real-Time adaptive filter and C) performing dimensionality reduction (from 70 to 2 features) and then classifying subjects using a supervised machine learning algorithm. The forecasting classification accuracy across 13 patients is 99.1% ± 0.004 and the area under the micro and macro ROC curve is 0.935 ± 0.036 and 0.940 ±0.034 as determined by 10-fold cross validation. The proposed approach enables a computationally efficient solution which could be implemented in a wearable device for preventing muscle injury.
Rodriguez-Manzano J, Miscourides N, Malpartida-Cardenas K, et al., 2019, Rapid detection of Klebsiella pneumoniae using an auto-calibrated ISFET-Array Lab-on-Chip platform
© 2019 IEEE. This paper presents the rapid detection of Klebsiella pneumonia, a major worldwide source and shuttle for antibiotic resistance, by a CMOS-based Lab-on-chip (LoC) platform. The LoC platform comprises a 64 × 200 ISFET array including a programmable gate for mismatch calibration. Calibration takes place on a pixel per pixel basis, with the array carrying-out simultaneous calibration and readout. The LoC platform is used to detect the pH changes occurring during DNA amplification and is demonstrated for the rapid detection of Klebsiella pneumoniae using isothermal molecular methods. Nucleic acids from the bacterial strain have been isolated from pure microbiological cultures and are detected in under 10 minutes. Overall, the presented Lab-on-Chip platform paired with the molecular methods hold significant potential for the rapid detection of Klebsiella pneumoniae at the point-of-care.
Toumazou C, Baig Mirza K, Rodriguez Manzano J, 2019, Molecule detection using aptamer nucleic acid duplex, WO2019138255A1
The present invention relates to the detection of molecules of a target type in a sample. A method of detecting molecules of a target type in a sample comprises providing an aptamer-nucleic acid duplex, contacting the sample with the aptamer-nucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of the target type in the sample, amplifying any dissociated nucleic acid and detecting any amplified nucleic acid. The method further comprises using the detected result to indicate the presence of molecules of the target type and/or quantify an amount of molecules of the target type. Also provided is a system for detecting molecules of a target type in a sample.
Malpartida-Cardenas K, Miscourides N, Rodriguez-Manzano J, et al., 2019, Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Early and accurate diagnosis of malaria and drug-resistance is essential to effective disease management. Available rapid malaria diagnostic tests present limitations in analytical sensitivity, drug-resistant testing and/or quantification. Conversely, diagnostic methods based on nucleic acid amplification stepped forwards owing to their high sensitivity, specificity and robustness. Nevertheless, these methods commonly rely on optical measurements and complex instrumentation which limit their applicability in resource-poor, point-of-care settings. This paper reports the specific, quantitative and fully-electronic detection of <jats:italic>Plas-modium falciparum</jats:italic>, the predominant malaria-causing parasite worldwide, using a Lab-on-Chip platform developed in-house. Furthermore, we demonstrate on-chip detection of C580Y, the most prevalent single-nucleotide polymorphism associated to artemisinin-resistant malaria. Real-time non-optical DNA sensing is facilitated using Ion-Sensitive Field-Effect Transistors, fabricated in unmodified complementary metal-oxide-semiconductor technology, coupled with loop-mediated isothermal amplification. This work holds significant potential for the development of a fully portable and quantitative malaria diagnostic that can be used as a rapid point-of-care test.</jats:p>
Moniri A, Rodriguez-Manzano J, Malpartida-Cardenas K, et al., 2019, Framework for DNA quantification and outlier detection using multidimensional standard curves, Analytical Chemistry, Vol: 91, Pages: 7426-7434, ISSN: 0003-2700
Real-time PCR is a highly sensitive and powerful technology for the quantification of DNA and has become the method of choice in microbiology, bioengineering, and molecular biology. Currently, the analysis of real-time PCR data is hampered by only considering a single feature of the amplification profile to generate a standard curve. The current “gold standard” is the cycle-threshold (Ct) method which is known to provide poor quantification under inconsistent reaction efficiencies. Multiple single-feature methods have been developed to overcome the limitations of the Ct method; however, there is an unexplored area of combining multiple features in order to benefit from their joint information. Here, we propose a novel framework that combines existing standard curve methods into a multidimensional standard curve. This is achieved by considering multiple features together such that each amplification curve is viewed as a point in a multidimensional space. Contrary to only considering a single-feature, in the multidimensional space, data points do not fall exactly on the standard curve, which enables a similarity measure between amplification curves based on distances between data points. We show that this framework expands the capabilities of standard curves in order to optimize quantification performance, provide a measure of how suitable an amplification curve is for a standard, and thus automatically detect outliers and increase the reliability of quantification. Our aim is to provide an affordable solution to enhance existing diagnostic settings through maximizing the amount of information extracted from conventional instruments.
Ming D, Rawson T, Sangkaew S, et al., 2019, Connectivity of rapid-testing diagnostics and surveillance of infectious diseases, Bulletin of the World Health Organization, Vol: 97, Pages: 242-244, ISSN: 0042-9686
The World Health Organization (WHO) developed the ASSURED criteria to describe the ideal characteristics for point-of-care testing in low-resource settings: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable.1 These standards describe. Over the last decade, widespread adoption of point-of-care testing has led to significant changes in clinical decision-making processes. The development of compact molecular diagnostics, such as the GeneXpert® platform, have enabled short turnaround times and allowed profiling of antimicrobial resistance. Although modern assays have increased operational requirements, many devices are robust and can be operated within communities with minimal training. These new generation of rapid tests have bypassed barriers to care and enabled treatment to take place independently from central facilities. Here we describe the importance of connectivity, the automatic capture and sharing of patient healthcare data from testing, in the adoption and roll-out of rapid testing.
Yu L-S, Rodriguez-Manzano J, Malpartida-Cardenas K, et al., 2019, Rapid and sensitive detection of azole-resistant Aspergillus fumigatus by tandem-repeat loop-mediated isothermal amplification, Journal of Molecular Diagnostics, Vol: 21, Pages: 286-295, ISSN: 1525-1578
Invasive human fungal infections caused by multi-azole resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multi-azole resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single nucleotide polymorphism, is associated with the occurrence of multi-azole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.
Ming D, Rawson T, Sangkaew S, et al., 2019, Connectivity of rapid-testing diagnostics and surveillance of infectious diseases (vol 97, pg 244, 2019), BULLETIN OF THE WORLD HEALTH ORGANIZATION, Vol: 97, ISSN: 0042-9686
Rodriguez-Manzano J, Moniri A, Malpartida-Cardenas K, et al., 2019, Simultaneous single-channel multiplexing and quantification of carbapenem-resistant genes using multidimensional standard curves, Analytical Chemistry, Vol: 91, Pages: 2013-2020, ISSN: 0003-2700
Multiplexing and quantification of nucleic acids, both have, in their own right, significant and extensive use in biomedical related fields. Currently, the ability to detect several nucleic acid targets in a single-reaction scales linearly with the number of targets; an expensive and time-consuming feat. Here, we propose a new methodology based on multidimensional standard curves that extends the use of real-time PCR data obtained by common qPCR instruments. By applying this novel method-ology, we achieve simultaneous single-channel multiplexing and enhanced quantification of multiple targets using only real-time amplification data. This is obtained without the need of fluorescent probes, agarose gels, melting curves or sequencing analysis. Given the importance and demand for tackling challenges in antimicrobial resistance, the proposed method is ap-plied to four of the most prominent carbapenem-resistant genes: blaOXA-48, blaNDM, blaVIM and blaKPC, which account for 97% of the UK's reported carbapenemase-producing Enterobacteriaceae.
Ismagilov RF, Rodriguez Manzano J, Karymov M, et al., 2018, Devices and methods for direct visual detection and readout of single nucleic acid molecules, WO2017079696A1
The present invention relates to methods, computer readable medium and systems for detecting and counting single nucleic acid molecules confined in nanoliter volumes using an unmodified camera, such as a cell phone camera. In particular, it identifies colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones. The invention further provides an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users.
Malpartida-Cardenas K, Rodriguez-Manzano J, Yu L-S, et al., 2018, Allele-specific isothermal amplification method using novel unmodified self-stabilizing competitive primers, Analytical Chemistry, Vol: 90, Pages: 11972-11980, ISSN: 0003-2700
Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance.
Miscourides N, Yu L-S, Rodriguez-Manzano J, et al., 2018, A 12.8 k current-mode velocity-saturation ISFET array for on-chip real-time DNA detection, IEEE Transactions on Biomedical Circuits and Systems, Vol: 12, Pages: 1202-1214, ISSN: 1932-4545
This paper presents a large-scale CMOS chemical-sensing array operating in current mode for real-time ion imaging and detection of DNA amplification. We show that the current-mode operation of ion-sensitive field-effect transistors in velocity saturation devices can be exploited to achieve an almost perfect linearity in their input-output characteristics (pH-current), which are aligned with the continuous scaling trend of transistors in CMOS. The array is implemented in a 0.35-m process and includes 12.8 k sensors configured in a 2T per pixel topology. We characterize the array by taking into account nonideal effects observed with floating gate devices, such as increased pixel mismatch due to trapped charge and attenuation of the input signal due to the passivation capacitance, and show that the selected biasing regime allows for a sufficiently large linear range that ensures a linear pH to current despite the increased mismatch. The proposed system achieves a sensitivity of 1.03 A/pH with a pH resolution of 0.101 pH and is suitable for the real-time detection of the NDM carbapenemase gene in E. Coli using a loop-mediated isothermal amplification.
Rodriguez-Manzano J, Moniri A, Malpartida-Cardenas K, et al., 2018, Simultaneous single-channel multiplex and quantification of carbapenem-resistant genes using multidimensional standard curves
<jats:title>ABSTRACT</jats:title><jats:p>Multiplexing and absolute quantification of nucleic acids, both have, in their own right, significant and extensive use in biomedical related fields, especially in point-of-care applications. Currently, the ability to detect several nucleic acid targets in a single-reaction scales linearly with the number of targets; an expensive and time-consuming feat. Here, we propose a new methodology based on multidimensional standard curves that extends the use of real-time PCR data obtained by common qPCR instruments. By applying this novel methodology, we achieve simultaneous single-channel multiplexing and enhanced quantification of multiple targets using only real-time amplification data. This is obtained without the need of fluorescent probes, agarose gels, melting curves or sequencing analysis. Given the importance and demand for tackling challenges in antimicrobial resistance, the proposed method is applied to the four most prominent carbapenem-resistant genes: <jats:italic>bla</jats:italic><jats:sub>OXA-48</jats:sub>, <jats:italic>bla</jats:italic><jats:sub>NDM</jats:sub>, <jats:italic>bla</jats:italic><jats:sub>VIM</jats:sub> and <jats:italic>bla</jats:italic><jats:sub>KPC</jats:sub>, which account for 97% of the UK’s reported carbapenemase-producing Enterobacteriaceae.</jats:p>
Rodriguez-Manzano J, Ying Chia P, Wen Yeo T, et al., 2018, Improving Dengue diagnostics and management through innovative technology, Current Infectious Disease Reports, Vol: 20, ISSN: 1534-3146
Purpose of Review:Dengue continues to be a major global public health threat. Symptomatic infections can cause a spectrum of disease ranging from a mild febrile illness to severe and potentially life-threatening manifestations. Management relies on supportive treatment with careful fluid replacement. The purpose of this review is to define the unmet needs and challenges in current dengue diagnostics and patient monitoring and outline potential novel technologies to address these needs.Recent Findings:There have been recent advances in molecular and point-of-care (POC) diagnostics as well as technologies including wireless communication, low-power microelectronics, and wearable sensors that have opened up new possibilities for management, clinical monitoring, and real-time surveillance of dengue.Summary:Novel platforms utilizing innovative technologies for POC dengue diagnostics and wearable patient monitors have the potential to revolutionize dengue surveillance, outbreak response, and management at population and individual levels. Validation studies of these technologies are urgently required in dengue-endemic areas.
Rodriguez-Manzano J, Chia PY, Yeo TW, et al., 2018, Improving Dengue Diagnostics and Management Through Innovative Technology (vol 20, 25, 2018), CURRENT INFECTIOUS DISEASE REPORTS, Vol: 20, ISSN: 1523-3847
Moniri A, Rodriguez-Manzano J, Georgiou P, 2018, A framework for analysis of real-time nucleic acid amplification data using novel multidimensional standard curves
<jats:title>ABSTRACT</jats:title><jats:p>Research into improving methods for absolute quantification of nucleic acids using standard curves has plateaued despite its positive, far-reaching impact on biomedical applications and clinical diagnostics. Currently, the mathematics involved in this mature area is restricted by the simplicity of conventional standard curves such as the gold standard cycle-threshold (<jats:italic>C<jats:sub>t</jats:sub></jats:italic>) method. Here, we propose a novel framework that expands current methods into multidimensional space and opens the door for more complex mathematical techniques, signal processing and machine learning to be implemented. The heart of this work revolves around two new concepts: the multidimensional standard curve and its home - the feature space. This work has been validated using phage lambda DNA and standard qPCR instruments. We show that the capabilities of standard curves can be extended in order to simultaneously: enhance absolute quantification, detect outliers and provide insights into the intersection between molecular biology and amplification data. This work and its vision aims to maximise the information extracted from amplification data using current instruments without increasing the cost or complexity of existing diagnostic settings.</jats:p>
Abdulwahab M, Moser N, Rodriguez Manzano J, et al., A CMOS Bio-Chip combining pH Sensing, Temperature Regulation and Electric Field Generation for DNA Detection and Manipulation, 2018 IEEE International Symposium on Circuits and Systems (ISCAS), ISSN: 2379-447X
Au A, Moser N, Rodriguez Manzano J, et al., Live demonstration: a mobile diagnostic system for rapid detection and tracking of infectious diseases, 2018 IEEE International Symposium on Circuits and Systems (ISCAS), ISSN: 2379-447X
Moser N, Rodriguez-Manzano J, Lande TS, et al., 2018, A Scalable ISFET Sensing and Memory Array With Sensor Auto-Calibration for On-Chip Real-Time DNA Detection, IEEE TRANSACTIONS ON BIOMEDICAL CIRCUITS AND SYSTEMS, Vol: 12, Pages: 390-401, ISSN: 1932-4545
Fernandez-Cassi X, Timoneda N, Martinez-Puchol S, et al., 2018, Metagenomics for the study of viruses in urban sewage as a tool for public health surveillance, SCIENCE OF THE TOTAL ENVIRONMENT, Vol: 618, Pages: 870-880, ISSN: 0048-9697
Moser N, Rodriguez-Manzano J, Yu L-S, et al., 2017, Live Demonstration: A CMOS-Based ISFET Array for Rapid Diagnosis of the Zika Virus, IEEE International Symposium on Circuits and Systems (ISCAS) 2017, ISSN: 2379-447X
We demonstrate a diagnostics platform which integrates an ISFET array and a temperature control loop for isothermal DNA detection. The controller maintains a temperature of 63◦C to perform nucleic acid amplification which is detected by the on-chip sensors. The 32x32 ISFET array is first calibrated to cancel trapped charge and then measures the change in the pH of the reaction. The sensor data is sent to a microcontroller and the reaction is monitored in real-time using a MATLAB interface. Experiments confirm a change of 0.9 pH when tested for the presence of RNA associated with the Zika virus.
Ismagilov RF, Rodriguez Manzano J, Sun B, et al., 2017, Enhanced nucleic acid identification and detection, WO2015058008A3
The present invention relates to assays, including amplification assays, conducted in the presence of modulators. These assays can be used to detect the presence of particular nucleic acid sequences. In particular, these assays can allow for genotyping or other genetic analysis.
Ismagilov RF, Begolo S, Rodriguez Manzano J, 2016, Devices and Methods for Autonomous Measurements, WO2016105508A3
Disclosed herein are devices and methods for storing, processing, preparing and/or analyzing samples. The inventions herein also relate to strategies and methods for automating device operations and for combining multiple devices in an integrated platform.
Rodriguez-Manzano J, Karymov MA, Begolo S, et al., 2016, Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones, ACS NANO, Vol: 10, Pages: 3102-3113, ISSN: 1936-0851
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