| 1. |
- Ardabili, Sahar, et al.
(författare)
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Dean flow-coupled inertial focusing for ultra-high-throughput particle filtration
- 2010
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Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 1586-1588
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Konferensbidrag (refereegranskat)abstract
- Particle manipulation represents an important and fundamental step prior to counting, sorting and detecting bio-particles. In this study, we report dean-coupled inertial focusing of particles in flows through a single curve microchannel at extremely high channel Reynold numbers (∼325). We found the lateral particle focusing position, xf to be fixed and largely independent of radius of curvature and whether particles are pre-focused (at equilibrium) entering the curvature or randomly distributed. Finally, using a single inlet, u-shaped, microchannel we demonstrate filtration of 10μm particles from 2 μm particles at throughputs several orders of magnitude higher than previously shown.
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| 2. |
- Chinnasamy, Thiruppathiraja, et al.
(författare)
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A lateral flow paper microarray for rapid allergy point of care diagnostics
- 2014
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Ingår i: The Analyst. - : Royal Society of Chemistry. - 0003-2654 .- 1364-5528. ; 139:10, s. 2348-2354
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Tidskriftsartikel (refereegranskat)abstract
- There is a growing need for multiplexed specific IgE tests that can accurately evaluate patient sensitization profiles. However, currently available commercial tests are either single/low-plexed or require sophisticated instrumentation at considerable cost per assay. Here, we present a novel convenient lateral flow microarray-based device that employs a novel dual labelled gold nanoparticle-strategy for rapid and sensitive detection of a panel of 15 specific IgE responses in 35 clinical serum samples. Each gold nanoparticle was conjugated to an optimized ratio of HRP and anti-IgE, allowing significant enzymatic amplification to improve the sensitivity of the assay as compared to commercially available detection reagents. The mean inter-assay variability of the developed LFM assay was 12% CV, and analysis of a cohort of clinical samples (n = 35) revealed good general agreement with ImmunoCAP, yet with a varying performance among allergens (AUC = [0.54-0.88], threshold 1 kU). Due to the rapid and simple procedure, inexpensive materials and read-out by means of a consumer flatbed scanner, the presented assay may provide an interesting low-cost alternative to existing multiplexed methods when thresholds > 1 kU are acceptable.
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| 3. |
- Chinnasamy, Thiruppathiraja, et al.
(författare)
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Point-of-Care Vertical Flow Allergen Microarray Assay : Proof of Concept
- 2014
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 60:9, s. 1209-1216
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of < 10 min before imaging and data analysis. METHOD: Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of <= 10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS: A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was < 14%. The observed concordance with a clinical assay, Immuno-CAP, was R-2 = 0.89 (n = 31). CONCLUSIONS: In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.
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| 4. |
- Chinnasamy, Thiruppathiraja, et al.
(författare)
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Towards paper-based point of care affinity proteomics
- 2014
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - : Chemical and Biological Microsystems Society. - 9780979806476 ; , s. 64-66
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Konferensbidrag (refereegranskat)abstract
- Affinity-based methods such as protein microarrays have come to complement conventional mass/charge-based techniques for proteomic characterization of biological samples. Simultaneous measurement of hundreds or even thousands of serum biomarkers such as antibodies, antigens and other proteins may greatly improve the diagnostic accuracy in a variety of conditions such as autoimmune disorders, infections and several cancers. However, today's technologies for affinity proteomics are cumbersome, require expensive equipment and skilled operators. Here, we present two novel paper-based techniques developed in our lab that aim to bridge the gap between highly multiplexed affinity proteomics and point of care testing.
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| 5. |
- Dias, Jorge, et al.
(författare)
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Rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis
- 2018
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Ingår i: Journal of Visualized Experiments. - : Journal of Visualized Experiments. - 1940-087X. ; 2018:133
-
Tidskriftsartikel (refereegranskat)abstract
- The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.
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| 6. |
- Dias, Jorge T., et al.
(författare)
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Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays
- 2016
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 8:15, s. 8195-8201
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Tidskriftsartikel (refereegranskat)abstract
- Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 10(3)-fold (5 pg mL(-1)) lower than when no ultrasound was applied (50 ng mL(-1)) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.
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| 7. |
- Dias, Jorge T, et al.
(författare)
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Rapid signal enhancement method for nanoprobe-based biosensing
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing < 10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.
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| 8. |
- Dias, Jorge T., et al.
(författare)
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Rapid signal enhancement method for nanoprobe-based biosensing (vol 7, 2017)
- 2018
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- In the Methods section of this Article references 18 to 22 are incorrectly cited. The correct references were omitted from the reference list and appear below as references 1-5. References 18 to 22 are correctly cited in Introduction and Results and Discussion sections. "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.18." should read: "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.1." "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.19." should read: "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.2" "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych20." should read: "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych3." "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.21." should read: "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.4." "Microarrays were prepared as previously reported by our group22." should read: "Microarrays were prepared as previously reported by our group5.
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| 9. |
- Eklundh, A., et al.
(författare)
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Etiology of Clinical Community-Acquired Pneumonia in Swedish Children Aged 1-59 Months with High Pneumococcal Vaccine Coverage-The TREND Study
- 2021
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Ingår i: Vaccines. - : MDPI AG. - 2076-393X. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- (1) Immunization with pneumococcal conjugate vaccines has decreased the burden of community-acquired pneumonia (CAP) in children and likely led to a shift in CAP etiology. (2) The Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) enrolled children 1-59 months with clinical CAP according to the World Health Organization (WHO) criteria at Sachs' Children and Youth Hospital, Stockholm, Sweden. Children with rhonchi and indrawing underwent "bronchodilator challenge". C-reactive protein and nasopharyngeal PCR detecting 20 respiratory pathogens, were collected from all children. Etiology was defined according to an a priori defined algorithm based on microbiological, biochemical, and radiological findings. (3) Of 327 enrolled children, 107 (32%) required hospitalization; 91 (28%) received antibiotic treatment; 77 (24%) had a chest X-ray performed; and 60 (18%) responded to bronchodilator challenge. 243 (74%) episodes were classified as viral, 11 (3%) as mixed viral-bacterial, five (2%) as bacterial, two (0.6%) as atypical bacterial and 66 (20%) as undetermined etiology. After exclusion of children responding to bronchodilator challenge, the proportion of bacterial and mixed viral-bacterial etiology was 1% and 4%, respectively. (4) The novel TREND etiology algorithm classified the majority of clinical CAP episodes as of viral etiology, whereas bacterial etiology was uncommon. Defining CAP in children <5 years is challenging, and the WHO definition of clinical CAP is not suitable for use in children immunized with pneumococcal conjugate vaccines.
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| 10. |
- Gantelius, Jesper, et al.
(författare)
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A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays
- 2011
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 12:11, s. 7748-7759
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Tidskriftsartikel (refereegranskat)abstract
- Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.
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| 11. |
- Gantelius, Jesper, et al.
(författare)
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A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 82:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.
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| 12. |
- Gantelius, Jesper, et al.
(författare)
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A ten-minute high density lateral flow protein microarray assay
- 2011
-
Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011. - 9781618395955 ; , s. 1176-1178
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Konferensbidrag (refereegranskat)abstract
- Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<50ng/ml) determination of antigen specific antibodies in less than ten minutes total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy (AUC=99.4%) was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies.
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| 13. |
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| 14. |
- Gantelius, Jesper, et al.
(författare)
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Improved sensitivity on an allergen lateral flow microarray by means of dendritic amplification
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806421 ; , s. 1136-1137
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Konferensbidrag (refereegranskat)abstract
- Recently, paper-based substrates have been proposed as an alternative to commonly used activated glass slides for microarray patterning[1], used in conjunction with capillary driven lateral flow of sample and detection reagents through the membrane. While fluorescent detection reagents may be employed to achieve high sensitivity, gold nanoparticles can also be used to allow readout by means of common table top scanners or digital cameras. Here, we demonstrate first results from employing a dendritic, or layer-by-layer, amplification approach for a high-density lateral flow allergen protein microarray, indicating that substantially increased sensitivity can be achieved with very modest increase of assay handling and time requirements.
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| 15. |
- Gantelius, Jesper, et al.
(författare)
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Magnetic bead-based detection of autoimmune responses using protein microarrays.
- 2009
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Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784. ; 26, s. 269-276
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.
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| 16. |
- Gantelius, Jesper, 1980-
(författare)
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Novel diagnostic microarray assay formats towards comprehensive on-site analysis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.
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| 17. |
- Gaudenzi, Giulia, et al.
(författare)
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Point-of-Care Approaches for Meningitis Diagnosis in a Low-Resource Setting (Southwestern Uganda) : Observational Cohort Study Protocol of the "PI-POC" Trial
- 2020
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Ingår i: Journal of Medical Internet Research. - : JMIR Publications Inc.. - 1438-8871. ; 22:11
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Tidskriftsartikel (refereegranskat)abstract
- Background: A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. Objective: The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. Methods: The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid-based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. Results: A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. Conclusions: The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment.
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| 18. |
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| 19. |
- Lama, Lara, 1990-, et al.
(författare)
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A lateral flow cardiac troponin-T assay with colorimetric signal enhancement
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Cardiac troponin T (cTnT) is a biomarker for heart muscle damage such as in acute myocardial infarction (AMI). Its rapid assessment is needed to detect changes in the cTnT levels in blood for a quicker diagnosis of AMI. The sensitivity limit required to detect elevated levels of cTnT is 10 pg/mL, where the levels in the healthy population are 0.5-10 pg/mL. In this paper the detection of cardiac troponin T with a point-of-care lateral flow assay was investigated for the rapid diagnosis of AMI. It was studied by using different gold nanoparticle conjugation methods and colorimetric signal enhancement of detection gold nanoparticle conjugates could increase the sensitivity of a troponin T lateral flow microarray assay. The results indicate the same sensitivity range for the detection with gold nanoparticles functionalized with antibody by two different methods, and that the troponin T sandwich pair used might be essential for achieving a higher sensitivity. The signal enhancement increased the intensity signal of the detected cTnT on the array. The limit of detection of the assay changed from 10 μg/mL to 1 μg/mL for one conjugation method after signal enhancement but remained unchanged at 1 μg/mL for the other method.
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| 20. |
- Lama, Lara, 1990-
(författare)
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Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins.Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate false positive signals. Paper I shows improved assay sensitivity in a multiplexed vertical flow assay by the application of ultrasonic energy to the gold nanoparticles functionalized with detection antibodies. The ultrasonication of the antibody conjugated gold nanoparticles resulted in a 10 000 fold increase in sensitivity in a 3-plex assay. COMSOL Multiphysics was used to simulate the acoustical energy of the probe used in Paper I for obtaining an indication of the size and direction of the forces acting upon the functionalized gold nanoparticles.In Paper II, it was studied if different gold nanoparticle conjugation methods and colorimetric signal enhancement of the gold nanoparticle conjugates could influence the sensitivity of a paper-based lateral flow microarray assay, targeting cardiac troponin T for the rapid diagnostics of acute myocardial infarction.Ultrasonication and signal enhancement of the detection gold nanoparticles has the potential of improving the sensitivity of paper based assays and expanding their potential future applications.
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| 21. |
- Leblanc, Neil, et al.
(författare)
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Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.
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| 22. |
- Lindström, Sara, et al.
(författare)
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PCR amplification and genetic analysis in a microwell cell cultivation chip
- 2008
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Ingår i: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference. - : Chemical and Biological Microsystems Society. ; , s. 576-578
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Konferensbidrag (refereegranskat)abstract
- We present a method for long-term single cell/clone cultivation followed by cell lysis, DNA amplification and detection of PCR product in a chip containing 672 individual microwells. By performing all steps on-chip in microwells, the proliferation and cell morphology of every single cell or clone can be linked to its genetic information. In this study two mammalian cell lines (mutated A431 vs. wild type U-2 OS) were used as a model system for mutation screening in the p53 gene. The presented method could improve the sensitivity in mutation frequency analysis of heterogeneous tumor samples.
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| 23. |
- Nybond, Susanna, et al.
(författare)
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Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray.
- 2019
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 411:4, s. 813-822
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Tidskriftsartikel (refereegranskat)abstract
- Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.
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| 24. |
- Pati, Falguni, et al.
(författare)
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3D Bioprinting of Tissue/Organ Models
- 2016
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Ingår i: Angewandte Chemie International Edition. - : John Wiley & Sons. - 1433-7851 .- 1521-3773. ; 55:15, s. 4650-4665
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Forskningsöversikt (refereegranskat)abstract
- Invitro tissue/organ models are useful platforms that can facilitate systematic, repetitive, and quantitative investigations of drugs/chemicals. The primary objective when developing tissue/organ models is to reproduce physiologically relevant functions that typically require complex culture systems. Bioprinting offers exciting prospects for constructing 3D tissue/organ models, as it enables the reproducible, automated production of complex living tissues. Bioprinted tissues/organs may prove useful for screening novel compounds or predicting toxicity, as the spatial and chemical complexity inherent to native tissues/organs can be recreated. In this Review, we highlight the importance of developing 3D invitro tissue/organ models by 3D bioprinting techniques, characterization of these models for evaluating their resemblance to native tissue, and their application in the prioritization of lead candidates, toxicity testing, and as disease/tumor models.
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| 25. |
- Ramachandraiah, Harisha, et al.
(författare)
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Dean flow-coupled inertial focusing in curved channels
- 2014
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 8:3, s. 034117-
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Tidskriftsartikel (refereegranskat)abstract
- Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In inertial microfluidics, dominant inertial forces cause particles to move across streamlines and occupy equilibrium positions along the faces of walls in flows through straight micro channels. In this study, we systematically analyzed the addition of secondary Dean forces by introducing curvature and show how randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature. We found the lateral particle focusing position to be fixed and largely independent of radius of curvature and whether particles entering the curvature are pre-focused (at equilibrium) or randomly distributed. Unlike focusing in straight channels, where focusing typically is limited to channel cross-sections in the range of particle size to create single focusing point, we report here particle focusing in a large cross-section area (channel aspect ratio 1: 10). Furthermore, we describe a simple u-shaped curved channel, with single inlet and four outlets, for filtration applications. We demonstrate continuous focusing and filtration of 10 mu m particles (with > 90% filtration efficiency) from a suspension mixture at throughputs several orders of magnitude higher than flow through straight channels (volume flow rate of 4.25ml/min). Finally, as an example of high throughput cell processing application, white blood cells were continuously processed with a filtration efficiency of 78% with maintained high viability. We expect the study will aid in the fundamental understanding of flow through curved channels and open the door for the development of a whole set of bio-analytical applications.
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| 26. |
- Rasti, Reza, et al.
(författare)
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Health care workers' perceptions of point-of-care testing in a low-income country-A qualitative study in Southwestern Uganda
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 12:7
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Tidskriftsartikel (refereegranskat)abstract
- Background Point-of-care (POC) tests have become increasingly available and more widely used in recent years. They have been of particular importance to low-income settings, enabling them with clinical capacities that had previously been limited. POC testing programs hold a great potential for significant improvement in low-income health systems. However, as most POC tests are developed in high-income countries, disengagement between developers and end-users inhibit their full potential. This study explores perceptions of POC test end-users in a low-income setting, aiming to support the development of novel POC tests for low-income countries. Methods A qualitative study was conducted in Mbarara District, Southwestern Uganda, in October 2014. Fifty health care workers were included in seven focus groups, comprising midwives, laboratory technicians, clinical and medical officers, junior and senior nurses, and medical doctors. Discussions were audio-recorded and transcribed verbatim. Transcripts were coded through a data-driven approach for qualitative content analysis. Results Nineteen different POC tests were identified as currently being in use. While participants displayed being widely accustomed to and appreciative of the use of POC tests, they also assessed the use and characteristics of current tests as imperfect. An ideal POC test was characterized as being adapted to local conditions, thoughtfully implemented in the specific health system, and capable of improving the care of patients. Tests for specific medical conditions were requested. Opinions differed with regard to the ideal distribution of POC tests in the local health system. Conclusion POC tests are commonly used and greatly appreciated in this study setting. However, there are dissatisfactions with current POC tests and their use. To maximize benefit, stakeholders need to include end-user perspectives in the development and implementation of POC tests. Insights from this study will influence our ongoing efforts to develop POC tests that will be particularly usable in low-income settings.
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| 27. |
- Rasti, Reza, et al.
(författare)
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Point-of-care testing in a high-income country paediatric emergency department : a qualitative study in Sweden
- 2021
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Ingår i: BMJ Open. - : BMJ. - 2044-6055. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Objectives In many resource-limited health systems, point-of-care tests (POCTs) are the only means for clinical patient sample analyses. However, the speed and simplicity of POCTs also makes their use appealing to clinicians in high-income countries (HICs), despite greater laboratory accessibility. Although also part of the clinical routine in HICs, clinician perceptions of the utility of POCTs are relatively unknown in such settings as compared with others. In a Swedish paediatric emergency department (PED) where POCT use is routine, we aimed to characterise healthcare providers' perspectives on the clinical utility of POCTs and explore their implementation in the local setting; to discuss and compare such perspectives, to those reported in other settings; and finally, to gather requests for ideal novel POCTs. Design Qualitative focus group discussions study. A data-driven content analysis approach was used for analysis. Setting The PED of a secondary paediatric hospital in Stockholm, Sweden. Participants Twenty-four healthcare providers clinically active at the PED were enrolled in six focus groups. Results A range of POCTs was routinely used. The emerging theme Utility of our POCT use is double-edged illustrated the perceived utility of POCTs. While POCT services were considered to have clinical and social value, the local routine for their use was named to distract clinicians from the care for patients. Requests were made for ideal POCTs and their implementation. Conclusion Despite their clinical integration, deficient implementation routines limit the benefits of POCT services to this well-resourced paediatric clinic. As such deficiencies are shared with other settings, it is suggested that some characteristics of POCTs and of their utility are less related to resource level and more to policy deficiency. To address this, we propose the appointment of skilled laboratory personnel as ambassadors to hospital clinics offering POCT services, to ensure higher utility of such services.
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| 28. |
- Reu, Pedro, et al.
(författare)
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A 61% lighter cell culture dish to reduce plastic waste
- 2019
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution.
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| 29. |
- Reuterswärd, Philippa, et al.
(författare)
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An 8 minute colorimetric paper-based reverse phase vertical flow serum microarray for screening of hyper IgE syndrome
- 2015
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Ingår i: The Analyst. - : Royal Society of Chemistry. - 0003-2654 .- 1364-5528. ; 140:21, s. 7327-7334
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Tidskriftsartikel (refereegranskat)abstract
- Reverse phase microarrays are useful tools for affinity-based detection in hundreds of samples simultaneously. However, current methods typically require long assay times and fluorescent detection. Here we describe a paper-based Vertical Flow Microarray (VFM) assay as a rapid 8-minute colorimetric alternative for reverse phase microarray analysis. The VFM platform was optimized for detection of IgE with a detection limit of 1.9 μg mL-1 in whole serum. Optimized conditions were then used to screen 113 serum samples simultaneously for hyper IgE syndrome (hIgE), a rare primary immunodeficiency characterized by elevated levels of IgE. The same set of samples were then analysed with a conventional planar microarray with fluorescent detection for head-to-head testing. Both assays found elevated levels in three out of four hIgE patient samples, whereas no control samples displayed elevated levels in either method. The comparison experiments showed a good correlation between the two assays, as determined from a linear correlation study (Pearson's r = 0.76). Further, the assay-time reduction and reproducibility (intra assay CV = 12.4 ± 4.11%) demonstrate the applicability of the VFM platform for high throughput reverse phase screening.
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| 30. |
- Rhedin, S. A., et al.
(författare)
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Protocol Introducing a New Algorithm for Classification of Etiology in Studies on Pediatric Pneumonia: Protocol for the Trial of Respiratory Infections in Children for Enhanced Diagnostics Study
- 2019
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Ingår i: Journal of Medical Internet Research. - : JMIR Publications Inc.. - 1438-8871. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a need to better distinguish viral infections from antibiotic-requiring bacterial infections in children presenting with clinical community-acquired pneumonia (CAP) to assist health care workers in decision making and to improve the rational use of antibiotics. Objective: The overall aim of the Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) study is to improve the differential diagnosis of bacterial and viral etiologies in children aged below 5 years with clinical CAP, by evaluating myxovirus resistance protein A (MxA) as a biomarker for viral CAP and by evaluating an existing (multianalyte point-of-care antigen detection test system [mariPOC respi] ArcDia International Oy Ltd.) and a potential future point-of-care test for respiratory pathogens. Methods: Children aged 1 to 59 months with clinical CAP as well as healthy, hospital-based, asymptomatic controls will be included at a pediatric emergency hospital in Stockholm, Sweden. Blood (analyzed for MxA and C-reactive protein) and nasopharyngeal samples (analyzed with real-time polymerase chain reaction as the gold standard and antigen-based mariPOC respi test as well as saved for future analyses of a novel recombinase polymerase amplification-based point-of-care test for respiratory pathogens) will be collected. A newly developed algorithm for the classification of CAP etiology will be used as the reference standard. Results: A pilot study was performed from June to August 2017. The enrollment of study subjects started in November 2017. Results are expected by the end of 2019.Conclusions: The findings from the TREND study can be an important step to improve the management of children with clinical. © 2019 Journal of Medical Internet Research. All rights reserved.
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| 31. |
- Rhedin, Samuel, et al.
(författare)
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Myxovirus resistance protein A for discriminating between viral and bacterial lower respiratory tract infections in children- The TREND study
- 2022
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Ingår i: Clinical Microbiology and Infection. - : Elsevier BV. - 1198-743X .- 1469-0691. ; 28:9, s. 1251-1257
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Discriminating between viral and bacterial lower respiratory tract infection (LRTI) in children is challenging, leading to an excessive use of antibiotics. Myxovirus resistance protein A (MxA) is a promising biomarker for viral infections. The primary aim of the study was to assess differences in blood MxA levels between children with viral and bacterial LRTI. Secondary aims were to assess differences in blood MxA levels between children with viral LRTI and asymptomatic controls and to assess MxA levels in relation to different respiratory viruses. Methods: Children with LRTI were enrolled as cases at Sachs' Children and Youth Hospital, Stockholm, Sweden. Nasopharyngeal aspirates and blood samples for analysis of viral PCR, MxA, and C-reactive protein were systematically collected from all study subjects in addition to standard laboratory/radiology assessment. Aetiology was defined according to an algorithm based on laboratory and radiological findings. Asymptomatic children with minor surgical disease were enrolled as controls. Results: MxA levels were higher in children with viral LRTI (n = 242) as compared to both bacterial (n = 5) LRTI (p < 0.01, area under the curve (AUC) 0.90, 95% CI: 0.81 to 0.99), and controls (AUC 0.92, 95% CI: 0.88 to 0.95). In the subgroup of children with pneumonia diagnosis, a cutoff of MxA 430 mg/l discriminated between viral (n = 29) and bacterial (n = 4) aetiology with 93% (95% CI: 78-99%) sensi-tivity and 100% (95% CI: 51-100%) specificity (AUC 0.98, 95% CI: 0.94 to 1.00). The highest MxA levels were seen in cases PCR positive for influenza (median MxA 1699 mg/l, interquartile range: 732 to 2996) and respiratory syncytial virus (median MxA 1115 mg/l, interquartile range: 679 to 2489). Discussion: MxA accurately discriminated between viral and bacterial aetiology in children with LRTI, particularly in the group of children with pneumonia diagnosis, but the number of children with bacterial LRTI was low.
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| 32. |
- Rivas, Lourdes, et al.
(författare)
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A vertical flow paper-microarray assay with isothermal DNA amplification for detection of Neisseria meningitidis
- 2018
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Ingår i: Talanta. - : Elsevier. - 0039-9140 .- 1873-3573. ; 183, s. 192-200
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Tidskriftsartikel (refereegranskat)abstract
- Paper-based biosensors offer a promising technology to be used at the point of care, enabled by good performance, convenience and low-cost. In this article, we describe a colorimetric vertical-flow DNA microarray (DNA-VFM) that takes advantage of the screening capability of DNA microarrays in a paper format together with isothermal amplification by means of Recombinase Polymerase Amplification (RPA). Different assay parameters such as hybridization buffer, flow rate, printing buffer and capture probe concentration were optimized. A limit of detection (LOD) of 4.4 nM was achieved as determined by tabletop scanning. The DNA-VFM was applied as a proof of concept for detection of Neisseria meningitidis, a primary cause of bacterial meningitis. The LOD was determined to be between 38 and 2.1Å~106 copies/VFM assay, depending on the choice of DNA capture probes. The presented approach provides multiplex capabilities of DNA microarrays in a paper-based format for future point-of-care applications.
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| 33. |
- Ståhl, Patrik L., et al.
(författare)
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Visual DNA : Identification of DNA sequence variations by bead trapping
- 2007
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Ingår i: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 90, s. 741-745
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Tidskriftsartikel (refereegranskat)abstract
- In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.
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| 34. |
- Svedberg, Gustav, et al.
(författare)
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A printer-free, vertical flow based, colorimetric planar bead array for point of care applications
- 2015
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Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806483 ; , s. 945-947
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Konferensbidrag (refereegranskat)abstract
- We present a novel planar bead array that utilizes fluorescent colour coding, a convenient and quick bead immobilization technique, rapid pump-driven sample delivery and colorimetric readout enabling analysis of the array using an inexpensive USB microscope or smartphone camera. The array combines high multiplexing potential with a low assay run time and point of care amenability due to low equipment requirements.
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| 35. |
- Svedberg, Gustav, 1989-
(författare)
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Novel planar and particle-based microarrays for point-of-care diagnostics
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Point-of-care assays are easy-to-use, portable and inexpensive tests that canbe used to aid diagnostics by measuring levels of disease-specific moleculesin settings where access to advanced laboratory equipment and trainedpersonnel are limited, such as at the patient's bedside or in low resourceparts of developing countries. In order to achieve high multiplexingcapacities, such assays can be based on planar microarrays consisting ofspots immobilized on a flat surface or on particle-based microarrays basedon populations of encoded particles. The aim of the work presented in thisthesis is to develop new point-of-care amenable planar and particle-basedmicroarrays that allow for highly multiplexed assays while maintaining lowsample-to-result times, complexity and instrumentation requirements.Paper I demonstrates the use graphically encoded particles for colorimetricdetection of autoantibodies using a consumer-grade flatbed scanner. Fourgraphical characters on the surface of each particle allows for millions ofcodes and the use of gold nanoparticles as a detection label allows both thecode and the signal intensity to be read out in a single image.Paper II describes a signal enhancement method that increases thesensitivity of gold nanoparticle detection on planar microarrays. Using thismethod, detection of allergen-specific IgE can be carried out using aconsumer-grade flatbed scanner instead of a more expensive fluorescencescanner without sacrificing assay performance.Paper III demonstrates the use of an isothermal DNA amplification methodfor detection of adenoviral DNA on a paper-based microarray. Using anisothermal amplification method eliminates the need for a thermocycler,reducing the instrumentation required for such detection.Paper IV shows the use of solid-phase PCR to amplify bacterial DNA directlyon the surface of particles. This strategy reduces assay time by eliminatingthe need for separate amplification and hybridisation steps.
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| 36. |
- Svedberg, Gustav, et al.
(författare)
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Towards encoded particles for highly multiplexed colorimetric point of care autoantibody detection
- 2017
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 17:3, s. 549-556
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Tidskriftsartikel (refereegranskat)abstract
- Highly multiplexed point of care tests could improve diagnostic accuracy and differential diagnostic capacity in for instance emergency medicine and low resource environments. Available technology platforms for POC biomarker detection are typically simplex or low-plexed, whereas common lab-based microarray systems allow for the simultaneous detection of thousands of DNA or protein biomarkers. In this study, we demonstrate a novel suspension particle array platform that utilizes 900 mu m bricks for POC amenable colorimetric biomarker detection with an encoding capacity of over two million. Due to the mm-scale size, both the lithographic codes and colorimetric signals of individual particles can be visualized using a consumer grade office flatbed scanner, with a potential for simultaneous imaging of around 19000 particles per scan. The analytical sensitivity of the assay was determined to be 4 ng ml(-1) using an antibody model system. As a proof of concept, autoantibodies toward anoctamin 2 were detected in order to discriminate between multiple sclerosis plasma samples and healthy controls with p < 0.0001 and an inter-assay % CV of 9.44%.
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| 37. |
- Urrutia Iturritza, Miren, et al.
(författare)
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An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings
- 2024
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 15:6
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Tidskriftsartikel (refereegranskat)abstract
- Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.
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