Publications
105 results found
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.
Yu LS, Rodriguez-Manzano J, Moser N, et al., 2019, A CMOS-based lab-on-chip diagnostic system for rapid detection and worldwide monitoring of azole-resistant aspergillus fumigatus, Pages: 650-651
Rapid detection of azole-resistant Aspergillus fumigatus is urgently needed to prevent the spread of resistance caused by the misuse of drugs and fungicides. Current methods for detections of azole resistance are time-consuming and lab-dependent, which delays the treatment and promotes antifungal resistance in the long term. We report a newly developed rapid and portable diagnostics method via a combination of highly specific and sensitive isothermal amplification chemistries with an integrated Lab-on-Chip (LoC) platform. Compared to current diagnostic systems, our LoC platform has several advantages: (1) The system directly identifies the tandem repeat regions, which causes resistance to azole drugs in A. fumigatus; (2) Our LoC device is faster (detection time <30 min) than any current molecular-based commercial kits, which usually takes hours from samples to results; (3) Compared to conventional PCR instruments, our platform requires no optical constituents, which enables portability and low cost; (4) Our system can be controlled by a smartphone application (app) leveraging on cloud connectivity and global positioning system (GPS) to enable worldwide azole resistance surveillance [1] (Figure 1).
Rodriguez-Manzano J, Miscourides N, Malpartida-Cardenas K, et al., 2019, Rapid detection of <i>Klebsiella pneumoniae</i> using an auto-calibrated ISFET-array Lab-on-Chip platform, IEEE Biomedical Circuits and Systems Conference (BioCAS), Publisher: IEEE, ISSN: 2163-4025
Moser N, Keeble L, Rodriguez-Manzano J, et al., 2019, ISFET Arrays for Lab-on-Chip Technology : A Review, 26th IEEE International Conference on Electronics, Circuits and Systems (ICECS), Publisher: IEEE, Pages: 57-60
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- Citations: 14
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.
Ma D, Rodriguez-Manzano J, Lopez SDM, et al., 2018, Adapting ISFETs for Epigenetics: An Overview, IEEE TRANSACTIONS ON BIOMEDICAL CIRCUITS AND SYSTEMS, Vol: 12, Pages: 1186-1201, ISSN: 1932-4545
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, 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
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.
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>
Au A, Moser N, Rodriguez Manzano J, et al., 2018, 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
Abdulwahab M, Moser N, Rodriguez Manzano J, et al., 2018, 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
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
This paper presents a novel CMOS-based system-onchip with a 78 × 56 ion-sensitive field-effect transistor array using in-pixel quantization and compensation of sensor nonidealities. The pixel integrates sensing circuitry and memory cells to encode the ion concentration in time and store a calibration value per pixel. Temperature sensing pixels spread throughout the array allow temperature monitoring during the reaction. We describe the integration of the array as part of a lab-on-chip cartridge that plugs into a motherboard for power management, biasing, data acquisition, and temperature regulation. This forms a robust ion detection platform, which is demonstrated as a pH sensing system. We show that our calibration is able to perform readout with a linear spread of 0.3% and that the system exhibits a high pH sensitivity of 3.2 μs/pH. The complete system is shown to perform on-chip realtime DNA amplification and detection of lambda phage DNA by loop-mediated isothermal amplification.
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
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- Citations: 88
Malpartida-Cardenas K, Miscourides N, Yu LS, et al., 2018, A CMOS-based diagnostic system for detection of artemisinin-resistant malaria, Pages: 871-873
Rapid, sensitive and specific detection of single nucleotide polymorphisms (SNPs) at the point-of-care is urgently needed, specially related to the emergence of drug-resistant malaria. Here, we report a novel biosensing platform combining complementary metal-oxide semiconductor technology and loop-mediated isothermal amplification for the detection of SNPs related to artemisinin-resistant malaria. The proposed biosensing platform has shown rapid and specific detection of drug-resistant SNPs in less than 20 min, which is ideal for point-of-care diagnosis and treatment guidance.
Yu LS, Moser N, Au A, et al., 2018, A CMOS based lab-on-chip diagnostic system for rapid detection and serotyping of the dengue virus, Pages: 298-300
Dengue virus outbreaks pose an ongoing threat to public health. Here, we report an innovative point-of-care diagnostic device, capable of simultaneous dengue detection, serotyping and real-time reporting. Our platform comprised an ISFET-based system, with 78x56 ISFET array permitting DNA/RNA amplification and detection, as well as an Android OS application on a smart phone to collect and report infections. Both synthetic and clinical sample validations confirm a change of pH when tested for the presence of dengue viral RNAs. These results show that our platform has the potential to accelerate the screening of 4 dengue serotype infections in order to provide rapid diagnostics and the real-time surveillance.
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
Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying 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. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.
Witters D, Sun B, Begolo S, et al., 2014, Digital biology and chemistry, Lab on a Chip, Vol: 14, Pages: 3225-3232, ISSN: 1473-0197
This account examines developments in "digital" biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide "analog-to-digital" signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in "digital" microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This "digitizing" offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more
Girones R, Carratala A, Calgua B, et al., 2014, Chlorine inactivation of hepatitis E virus and human adenovirus 2 in water, JOURNAL OF WATER AND HEALTH, Vol: 12, Pages: 436-442, ISSN: 1477-8920
Sun B, Rodriguez-Manzano J, Selck DA, et al., 2014, Measuring Fate and Rate of Single-Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 53, Pages: 8088-8092, ISSN: 1433-7851
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- Citations: 22
Rodriguez-Manzano J, Hundesa A, Calgua B, et al., 2014, Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish, FOOD AND ENVIRONMENTAL VIROLOGY, Vol: 6, Pages: 31-41, ISSN: 1867-0334
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- Citations: 23
Carratala A, Rusinol M, Rodriguez-Manzano J, et al., 2013, Environmental Effectors on the Inactivation of Human Adenoviruses in Water, FOOD AND ENVIRONMENTAL VIROLOGY, Vol: 5, Pages: 203-214, ISSN: 1867-0334
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- Citations: 22
Carratala A, Rodriguez-Manzano J, Hundesa A, et al., 2013, Effect of temperature and sunlight on the stability of human adenoviruses and MS2 as fecal contaminants on fresh produce surfaces, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, Vol: 164, Pages: 128-134, ISSN: 0168-1605
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- Citations: 22
Calgua B, Fumian T, Rusinol M, et al., 2013, Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas, WATER RESEARCH, Vol: 47, Pages: 2797-2810, ISSN: 0043-1354
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- Citations: 85
Calgua B, Rodriguez-Manzano J, Hundesa A, et al., 2013, New methods for the concentration of viruses from urban sewage using quantitative PCR, JOURNAL OF VIROLOGICAL METHODS, Vol: 187, Pages: 215-221, ISSN: 0166-0934
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- Citations: 77
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