Publications by authors named "Narayanan Madaboosi"

21 Publications

  • Page 1 of 1

Rolling Circle Amplification in Integrated Microsystems: An Uncut Gem toward Massively Multiplexed Pathogen Diagnostics and Genotyping.

Acc Chem Res 2021 Oct 12. Epub 2021 Oct 12.

Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 17165 Solna, Sweden.

ConspectusThe development of robust methods allowing the precise detection of specific nucleic acid sequences is of major societal relevance, paving the way for significant advances in biotechnology and biomedical engineering. These range from a better understanding of human disease at a molecular level, allowing the discovery and development of novel biopharmaceuticals and vaccines, to the improvement of biotechnological processes providing improved food quality and safety, efficient green fuels, and smart textiles. Among these applications, the significance of pathogen diagnostics as the main focus of this Account has become particularly clear during the recent SARS-CoV-2 pandemic. In this context, while RT-PCR is the gold standard method for unambiguous detection of genetic material from pathogens, other isothermal amplification alternatives circumventing rapid heating-cooling cycles up to ∼95 °C are appealing to facilitate the translation of the assay into point-of-care (PoC) analytical platforms. Furthermore, the possibility of routinely multiplexing the detection of tens to hundreds of target sequences with single base pair specificity, currently not met by state-of-the-art methods available in clinical laboratories, would be instrumental along the path to tackle emergent viral variants and antimicrobial resistance genes. Here, we advocate that padlock probes (PLPs), first reported by Nilsson et al. in 1994, coupled with rolling circle amplification (RCA), termed here as PLP-RCA, is an underexploited technology in current arena of isothermal nucleic acid amplification tests (NAATs) providing an unprecedented degree of multiplexing, specificity, versatility, and amenability to integration in miniaturized PoC platforms. Furthermore, the intrinsically digital amplification of PLP-RCA retains spatial information and opens new avenues in the exploration of pathogenesis with spatial multiomics analysis of infected cells and tissue.The Account starts by introducing PLP-RCA in a nutshell focusing individually on the three main assay steps, namely, (1) PLP design and ligation mechanism, (2) RCA after probe ligation, and (3) detection of the RCA products. Each subject is touched upon succinctly but with sufficient detail for the reader to appreciate some assay intricacies and degree of versatility depending on the analytical challenge at hand. After familiarizing the reader with the method, we discuss specific examples of research in our group and others using PLP-RCA for viral, bacterial, and fungal diagnostics in a variety of clinical contexts, including the genotyping of antibiotic resistance genes and viral subtyping. Then, we dissect key developments in the miniaturization and integration of PLP-RCA to minimize user input, maximize analysis throughput, and expedite the time to results, ultimately aiming at PoC applications. These developments include molecular enrichment for maximum sensitivity, spatial arrays to maximize analytical throughput, automation of liquid handling to streamline the analytical workflow in miniaturized devices, and seamless integration of signal transduction to translate RCA product titers (and ideally spatial information) into a readable output. Finally, we position PLP-RCA in the current landscape of NAATs and furnish a systematic Strengths, Weaknesses, Opportunities and Threats analysis to shine light upon unpolished edges to uncover the gem with potential for ubiquitous, precise, and unbiased pathogen diagnostics.
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http://dx.doi.org/10.1021/acs.accounts.1c00438DOI Listing
October 2021

Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes.

Anal Chem 2021 04 7;93(15):6169-6177. Epub 2021 Apr 7.

Department of Biosystems, Biosensors Group, KU Leuven, Willem De Croylaan 42, Heverlee B-3001, Belgium.

Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immuno-RCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.
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http://dx.doi.org/10.1021/acs.analchem.1c00172DOI Listing
April 2021

Electrochemical Genosensing of Based on Padlock Probes and Rolling Circle Amplification.

Sensors (Basel) 2021 Mar 3;21(5). Epub 2021 Mar 3.

Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.
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http://dx.doi.org/10.3390/s21051749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959471PMC
March 2021

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography.

Biosens Bioelectron 2020 Oct 26;166:112442. Epub 2020 Jul 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden. Electronic address:

Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of ~1 μm-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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http://dx.doi.org/10.1016/j.bios.2020.112442DOI Listing
October 2020

OPENchip: an on-chip in situ molecular profiling platform for gene expression analysis and oncogenic mutation detection in single circulating tumour cells.

Lab Chip 2020 03;20(5):912-922

Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, South Korea. and Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, South Korea and BK21+ Creative Research Engineer Development for IT, Seoul National University, Seoul, 08826, South Korea and Biomedical Research Institute, Seoul National University Hospital, Seoul, 03080, Republic of Korea and Institutes of Entrepreneurial BioConvergence, Seoul National University, Seoul, 08826, Republic of Korea.

Liquid biopsy holds promise towards practical implementation of personalized theranostics of cancer. In particular, circulating tumour cells (CTCs) can provide clinically actionable information that can be directly linked to prognosis or therapy decisions. In this study, gene expression patterns and genetic mutations in single CTCs are simultaneously analysed by strategically combining microfluidic technology and in situ molecular profiling technique. Towards this, the development and demonstration of the OPENchip (On-chip Post-processing ENabling chip) platform for single CTC analysis by epithelial CTC enrichment and subsequent in situ molecular profiling is reported. For in situ molecular profiling, padlock probes that identify specific desired targets to examine biomarkers of clinical relevance in cancer diagnostics were designed and used to create libraries of rolling circle amplification products. We characterize the OPENchip in terms of its capture efficiency and capture purity, and validate the probe design using different cell lines. By integrating the obtained results, molecular analyses of CTCs from metastatic breast cancer (HER2 (ERBB2) gene expression and PIK3CA mutations) and metastatic pancreatic cancer (KRAS gene mutations) patients were demonstrated without any off-chip processes. The results substantiate the potential implementation of early molecular detection of cancer through sequencing-free liquid biopsy.
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http://dx.doi.org/10.1039/c9lc01248fDOI Listing
March 2020

The sweet detection of rolling circle amplification: Glucose-based electrochemical genosensor for the detection of viral nucleic acid.

Biosens Bioelectron 2020 Mar 30;151:112002. Epub 2019 Dec 30.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Sweden. Electronic address:

Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.
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http://dx.doi.org/10.1016/j.bios.2019.112002DOI Listing
March 2020

Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses.

J Mol Diagn 2020 02 16;22(2):272-283. Epub 2019 Dec 16.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden. Electronic address:

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.
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http://dx.doi.org/10.1016/j.jmoldx.2019.10.014DOI Listing
February 2020

Top-Down Fabricated Silicon Nanowire Arrays for Field-Effect Detection of Prostate-Specific Antigen.

ACS Omega 2018 Aug 1;3(8):8471-8482. Epub 2018 Aug 1.

Department of Informatics and Microsystem Technology, University of Applied Sciences Kaiserslautern, Amerikastrasse 1, 66482 Zweibrücken, Germany.

Highly sensitive electrical detection of biomarkers for the early stage screening of cancer is desired for future, ultrafast diagnostic platforms. In the case of prostate cancer (PCa), the prostate-specific antigen (PSA) is of prime interest and its detection in combination with other PCa-relevant biomarkers in a multiplex approach is advised. Toward this goal, we demonstrate the label-free, potentiometric detection of PSA with silicon nanowire ion-sensitive field-effect transistor (Si NW-ISFET) arrays. To realize the field-effect detection, we utilized the DNA aptamer-receptors specific for PSA, which were covalently and site-specifically immobilized on Si NW-ISFETs. The platform was used for quantitative detection of PSA and the change in threshold voltage of the Si NW-ISEFTs was correlated with the concentration of PSA. Concentration-dependent measurements were done in a wide range of 1 pg/mL to 1 μg/mL, which covers the clinical range of interest. To confirm the PSA-DNA aptamer binding on the Si NW surfaces, a sandwich-immunoassay based on chemiluminescence was implemented. The electrical approach using the Si NW-ISFET platform shows a lower limit of detection and a wide dynamic range of the assay. In future, our platform should be utilized to detect multiple biomarkers in one assay to obtain more reliable information about cancer-related diseases.
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http://dx.doi.org/10.1021/acsomega.8b00990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644640PMC
August 2018

Correction to: Efficient DNA-assisted synthesis of trans-membrane gold nanowires.

Microsyst Nanoeng 2018 4;4. Epub 2018 Jun 4.

1Department of Micro and Nanosystems, KTH Royal Institute of Technology, Osquldas väg 10, Stockholm, 100 44 Sweden.

[This corrects the article DOI: 10.1038/micronano.2017.84.].
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http://dx.doi.org/10.1038/s41378-018-0012-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161504PMC
June 2018

A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses.

Sci Rep 2019 02 27;9(1):2872. Epub 2019 Feb 27.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE- 171 65, Solna, Sweden.

The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.
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http://dx.doi.org/10.1038/s41598-019-39854-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393471PMC
February 2019

Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range.

Biosens Bioelectron 2019 Mar 18;128:68-75. Epub 2018 Dec 18.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE-171 65 Solna, Sweden. Electronic address:

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 μL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.
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http://dx.doi.org/10.1016/j.bios.2018.12.004DOI Listing
March 2019

Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza.

Clin Chem 2018 12 26;64(12):1704-1712. Epub 2018 Sep 26.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden;

Background: Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed.

Methods: We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants-A(H1N1), A(H3N2), B/Yamagata, and B/Victoria-and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct.

Results: The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants.

Conclusions: We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.
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http://dx.doi.org/10.1373/clinchem.2018.292979DOI Listing
December 2018

A "Cell-Friendly" Window for the Interaction of Cells with Hyaluronic Acid/Poly-l-Lysine Multilayers.

Macromol Biosci 2018 02 12;18(2). Epub 2017 Dec 12.

Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Department Cellular Biotechnology & Biochips, Am Mühlenberg 13, 14476, Potsdam-Golm, Germany.

Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly-l-lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL) films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number "n" a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH-Fib, human embryonic kidney HEK-293, human bone cell line U-2-OS, Chinese hamster ovary CHO-K and mouse embryonic stem cells. All cells adhere and spread well in a narrow "cell-friendly" window identify in the range of n = 12-15. At n < 12, the film is inhomogeneous and at n > 15, the film is cell repellent for all cell lines. Cellular adhesion correlates with the mechanical properties of the films showing that softer films at higher "n" number exhibiting a significant decrease of the Young's modulus below 100 kPa are weakly adherent to cells. This trend cannot be reversed even by coating a strong cell-adhesive protein fibronectin onto the film. This indicates that mechanical cues plays a major role for cell behavior, also in respect to biochemical ones.
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http://dx.doi.org/10.1002/mabi.201700319DOI Listing
February 2018

Lab-on-chip systems for integrated bioanalyses.

Essays Biochem 2016 06;60(1):121-31

Instituto de Engenharia de Sistemas E Computadores-Microsistemas e Nanotecnologias (INESC MN) and IN-Institute of Nanoscience and Nanotechnology, Rua Alves Redol, 9, 1000-029 Lisbon, Portugal.

Biomolecular detection systems based on microfluidics are often called lab-on-chip systems. To fully benefit from the miniaturization resulting from microfluidics, one aims to develop 'from sample-to-answer' analytical systems, in which the input is a raw or minimally processed biological, food/feed or environmental sample and the output is a quantitative or qualitative assessment of one or more analytes of interest. In general, such systems will require the integration of several steps or operations to perform their function. This review will discuss these stages of operation, including fluidic handling, which assures that the desired fluid arrives at a specific location at the right time and under the appropriate flow conditions; molecular recognition, which allows the capture of specific analytes at precise locations on the chip; transduction of the molecular recognition event into a measurable signal; sample preparation upstream from analyte capture; and signal amplification procedures to increase sensitivity. Seamless integration of the different stages is required to achieve a point-of-care/point-of-use lab-on-chip device that allows analyte detection at the relevant sensitivity ranges, with a competitive analysis time and cost.
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http://dx.doi.org/10.1042/EBC20150013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986467PMC
June 2016

DNA aptamer-based sandwich microfluidic assays for dual quantification and multi-glycan profiling of cancer biomarkers.

Biosens Bioelectron 2016 May 19;79:313-9. Epub 2015 Dec 19.

Department of Electronic & Electrical Engineering, University of Bath, Bath BA2 7AY, United Kingdom. Electronic address:

Two novel sandwich-based immunoassays for prostate cancer (PCa) diagnosis are reported, in which the primary antibody for capture is replaced by a DNA aptamer. The assays, which can be performed in parallel, were developed in a microfluidic device and tested for the detection of free Prostate Specific Antigen (fPSA). A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of fPSA and its glycosylation levels. The use of aptamers enables a more reliable, selective and controlled sensing of the analyte. The dual approach provides sensitive detection of fPSA along with selective fPSA glycoprofiling, which is of significant importance in the diagnosis and prognosis of PCa, as tumor progression is associated with changes in fPSA glycosylation. With these approaches, we can potentially detect 0.5 ng/mL of fPSA and 3 ng/mL of glycosylated fPSA using Sambucus nigra (SNA) lectin, both within the relevant clinical range. The approach can be applied to a wide range of biomarkers, thus providing a good alternative to standard antibody-based immunoassays with significant impact in medical diagnosis and prognosis.
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http://dx.doi.org/10.1016/j.bios.2015.12.058DOI Listing
May 2016

A microfluidic immunoassay platform for the detection of free prostate specific antigen: a systematic and quantitative approach.

Analyst 2015 Jul;140(13):4423-33

INESC Microsistemas e Nanotecnologias (INESC MN) and IN - Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

As a leading cause of cancer-related deaths in men globally, prostate cancer (PCa) demands immense attention for theranostic purposes. There is an increasing need for the development of rapid, sensitive, economical, miniaturized and multiplexable assays. Towards this goal, we present a systematic approach for the optimisation of a microfluidic sandwich immunoassay, which can be applied as a generic biosensor platform for PCa detection. Prostate specific antigen (PSA) was used as the model biomarker, and its free form was captured using commercially available antibodies and detected using chemiluminescence, both in spiked buffer and matrix solutions. Along with the optimisation of surface chemistry and microfluidic parameters, we report a bio-affinity amplification strategy based on biotin-streptavidin chemistry to bring the limits of detection for free-PSA from 21.4 ng mL(-1) down to 2.7 ng mL(-1), within the clinically relevant range. An estimate of the surface coverage and simulations of the interactions taking place in the microfluidic biosensor during the assay are also presented. This novel platform using a simple passive adsorption-based bio-affinity strategy, when coupled with multiplexing and integrated detection, can serve as a promising point-of-care diagnostic tool for PCa.
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http://dx.doi.org/10.1039/c5an00364dDOI Listing
July 2015

Growth behaviour and mechanical properties of PLL/HA multilayer films studied by AFM.

Beilstein J Nanotechnol 2012 21;3:778-88. Epub 2012 Nov 21.

Stranski-Laboratorium, Department of Chemistry, TU Berlin, Strasse des 17. Juni 124, D-10623 Berlin, Germany.

Scanning- and colloidal-probe atomic force microscopy were used to study the mechanical properties of poly(L-lysine)/hyaluronan (PLL/HA)(n) films as a function of indentation velocity and the number of polymer deposition steps n. The film thickness was determined by two independent AFM-based methods: scratch-and-scan and newly developed full-indentation. The advantages and disadvantages of both methods are highlighted, and error minimization techniques in elasticity measurements are addressed. It was found that the film thickness increases linearly with the bilayer number n, ranging between 400 and 7500 nm for n = 12 and 96, respectively. The apparent Young's modulus E ranges between 15 and 40 kPa and does not depend on the indenter size or the film bilayer number n. Stress relaxation measurements show that PLL/HA films have a viscoelastic behaviour, regardless of their thickness. If indentation is performed several times at the same lateral position on the film, a viscous/plastic deformation takes place.
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http://dx.doi.org/10.3762/bjnano.3.87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3512127PMC
December 2012

Microfluidics as a tool to understand the build-up mechanism of exponential-like growing films.

Macromol Rapid Commun 2012 Oct 18;33(20):1775-9. Epub 2012 Jul 18.

Fraunhofer Institute of Biomedical Engineering, Am Mühlenberg 13, Potsdam-Golm, Germany.

Microfluidics is used here for the first time to efficiently tune the growth conditions for understanding the build-up mechanism of exponentially growing polyelectrolyte (PE) films. The velocity of PE supply and time of interaction can be successfully altered during the layer-by-layer assembly. Another advantage of this method is that the deposition of poly-L-lysine/hyaluronic acid (PLL/HA) films in microchannels can be monitored online by fluorescence microscopy. The study demonstrates that PE mass transport to the film surface and diffusion in the film are key parameters affecting PLL/HA film build-up. Increase of PE supply rate results in a change in the "transition" (exponential-to-linear growth) towards higher number of deposition steps, thus indicating a mass transport-mediated growth mechanism.
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http://dx.doi.org/10.1002/marc.201200353DOI Listing
October 2012

Control of cell adhesion by mechanical reinforcement of soft polyelectrolyte films with nanoparticles.

Langmuir 2012 May 30;28(18):7249-57. Epub 2012 Apr 30.

Fraunhofer Institut für Biomedizinische Technik, Am Mühlenberg 13, 14476 Potsdam-Golm, Germany.

Chemical cross-linking is the standard approach to tune the mechanical properties of polymer coatings for cell culture applications. Here we show that the elastic modulus of highly swollen polyelectrolyte films composed of poly(L-lysine) (PLL) and hyaluronic acid (HA) can be changed by more than 1 order of magnitude by addition of gold nanoparticles (AuNPs) in a one-step procedure. This hydrogel-nanoparticle architecture has great potential as a platform for advanced cell engineering application, for example remote release of drugs. As a first step toward utilization of such films for biomedical applications we identify the most favorable polymer/nanoparticle composition for optimized cell adhesion on the films. Using atomic force microscopy (AFM) we determine the following surface parameters that are relevant for cell adhesion, i.e., stiffness, roughness, and protein interactions. Optimized cell adhesion is observed for films with an elastic modulus of about 1 MPa and a surface roughness on the order of 30 nm. The analysis further shows that AuNPs are not incorporated in the HA/PLL bulk but form clusters on the film surface. Combined studies of the elastic modulus and surface topography indicate a cluster percolation threshold at a critical surface coverage above which the film stiffness drastically increases. In this context we also discuss changes in film thickness, material density and swelling ratio due to nanoparticle treatment.
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http://dx.doi.org/10.1021/la300635zDOI Listing
May 2012

Microfluidics meets soft layer-by-layer films: selective cell growth in 3D polymer architectures.

Lab Chip 2012 Apr 1;12(8):1434-6. Epub 2012 Mar 1.

Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, 14476 Potsdam-Golm, Germany.

We present here the micropatterns of layer-by-layer (LbL) assembled soft films generated using microfluidic platform that can be exploited for selective cell growth. Using this method, the issue of cell adhesion and spreading on soft LbL-derived films, and simultaneous utilisation of such unmodified soft films to exploit their reservoir properties are addressed. This also paves the way for extending the culture of cells to soft films and other demanding applications like triggered release of biomolecules.
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http://dx.doi.org/10.1039/c2lc40058hDOI Listing
April 2012

Anisotropic multicompartment micro- and nano-capsules produced via embedding into biocompatible PLL/HA films.

Chem Commun (Camb) 2011 Feb 24;47(7):2098-100. Epub 2010 Dec 24.

Max-Planck Institute of Colloids and Interfaces, 14424-Potsdam, Germany.

We present a novel strategy to fabricate anisotropic multicompartment Janus capsules by embedding larger containers into a soft poly-L-lysine/hyaluronic acid (PLL/HA) polymeric film, followed by adsorption of smaller containers on top of their unmasked surface. This research is also attractive for developing substrates for cell cultures.
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http://dx.doi.org/10.1039/c0cc04820hDOI Listing
February 2011
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