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Multiplex lateral flow detection and binary encoding enables a molecular colorimetric 7-segment display

December 2015
Lab on a Chip 16(2)

December 2015
16(2)

DOI:10.1039/C5LC01323B

Authors:

Jia Li

University of Freiburg

Joanne Macdonald

University of the Sunshine Coast

Multiplex expansion in point-of-care diagnostics usually requires a linear increase of premium commodities such as reagents or space. Here we demonstrate the power of binary and molecular encoding to compress device operations. We describe the first colorimetric 7-segment display on a paper-based biosensor, providing compact and intuitive read-outs for multiplex detections.

A) Design of single-plex LFDs: a dual-labeled analyte (RVFV DNA) was sandwiched between capture and detection antibodies due to antigen-antibody binding; AuNPs conjugated to the capture antibody (mouse anti-fluorescein antibody) enabled visualization of binding through the appearance of a red color at the test dot; rabbit antimouse antibody, which can directly bind the capture antibody (mouse anti-fluorescein), was deposited in parallel as a control. (B) Determination of detection limits for each single-plex assay: detection antibodies corresponding to the 3′ antigen of dual-labeled RVFV were deposited at the test dot; resultant assay color intensity (average and standard deviation of 4 individual tests) was quantified using ImageJ software and plotted against DNA concentration; a cut-off defined as three standard deviations above the average was used to determine the limit of detection for each assay. Experimental details are described in the ESI. †

…

Hepta-plex lateral flow detection results for combinations of seven antigen-antibody pairs. A: Positioning of detection antibodies deposited upon lateral flow strips. B: Hepta-plex lateral flow detection of 1 μM dual-labeled analyte alone (containing either 3′ BODIPY® FL, Cy®5, or dansyl), or combinations of dual-labeled analyte (1 μM) containing 3′ biotin, Cascade Blue®, digoxigenin, dintrophenyl, tetramethylrhodamine (TAMRA), and Texas Red® and either BODIPY® FL, Cy®5 or dansyl, as indicated. Assay was repeated three times and a representative photograph is shown. Experimental details are described in the ESI. †

…

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Featuring work from the Molecular Engineering Research

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Title: Multiplex lateral ﬂ ow detection and binary encoding

enables a molecular colorimetric 7-segment display

Binary and molecular encoding enables a digital-like 7 segment

display on a paper-based biosensor. The display operates without

batteries and wires, providing an e cient, compact, and intuitive

output for multiplexing applications.

As featured in:

See Jia Li and Joanne Macdonald,

Lab Chip, 2016, 16, 242.

www.rsc.org/loc

Lab on a Chip

COMMUNICATION

Cite this: Lab Chip,2016,16,242

Received 25th October 2015,

Accepted 24th November 2015

DOI: 10.1039/c5lc01323b

www.rsc.org/loc

Multiplex lateral flow detection and binary

encoding enables a molecular colorimetric

7-segment display†

Jia Li

and Joanne Macdonald*

Multiplex expansion in point-of-care diagnostics usually requires a

linear increase of premium commodities such as reagents or

space. Here we demonstrate the power of binary and molecular

encoding to compress device operations. We describe the first

colorimetric 7-segment display on a paper-based biosensor, pro-

viding compact and intuitive read-outs for multiplex detections.

Multiplexing is a critical parameter for increasing diagnostic

efficiency. The strategies that enable simultaneous analysis of

multiple samples are largely dependent on the underlying

diagnostic technology. Multiplex detection using enzyme-

linked immunosorbent assays, real-time polymerase chain

reactions, microarrays and/or bead-based methods (such as

the Bio-Rad Bio-Plex®Systems

and Luminex MagPix®

)

enable high-throughput and low volume processing, but

require non-portable equipment and trained personnel for

operation. For point-of-care settings, lateral flow devices

(LFDs) are ideal candidates, but few commercial devices

employ multiplexing. This is due to issues with specificity

and reproducibility, as well as expansion limits, as described

by Washburn, because the flow rate decreases with distance

from the conjugate pad.

Here we propose a novel solution

for expanding LFD multiplex detection that increases the effi-

ciency of detection without expanding the device dimensions

and consuming excess reagents. Our generic solution com-

presses multiplex LFD data by borrowing from computational

science and applying binary encoding to create signature pat-

terns of test dots. Thus, a sample is diagnosed based on the

set of test dots that appear on the device. By judicious

arrangement of test dots, we create a 7-segment LFD display

that simulates digital display of information for easy interpre-

tation by the end-user.

To develop a generic multiplex LFD, we first identified

twelve individual antigen–antibody single-plex assays able to

operate using a sandwich immunoassay format. In this format

an analyte is sandwiched between a capture and detection anti-

body via tagged antigens (Fig. 1A). Gold nanoparticles (AuNPs)

conjugated to the capture antibody transform analyte detection

into a colorimetric signal. We chose fluorescein and anti-

fluorescein as the common AuNP conjugated capture antibody,

and identified an additional twelve commercially available anti-

gen–antibody combinations that could potentially be used as

detection antibodies (Fig. 1B). The analyte was a single-

stranded DNA corresponding to a segment of the L gene of

Rift Valley fever virus (RVFV).

Importantly, we kept the RVFV

DNA analyte sequence identical in all tests (apart from the 3′

antigen labels) to eliminate any differences in behavior due

to subtle changes in nucleotide sequences.

In addition, use

of a dual-labeled synthetic analyte reduced the complexity of

interactions for our proof-of-concept demonstration (applied

implementation would utilize labeled oligonucleotide, anti-

body, or aptamer binding partners to capture an unlabeled

analyte). All twelve antigen–antibody pairs were effective as

detection entities in the single-plex LFDs (Fig. 1 & S1†), with

standard LFD sensitivities as low as 5 nM.

We note in partic-

ular the success of labels Alexa Fluor®488, Cascade Blue®,

Lucifer yellow, benzopyrene, BODIPY®FL, and dansyl, which

have not previously been applied in lateral flow detection.

After demonstrating successful operation of all twelve

detection antigen–antibody combinations in a single-plex

LFD, we then attempted to combine these into a multiplex

LFD array using a dot-matrix (3 ×4) format. For the arrange-

ment of detection antibodies on the membrane, we hypothe-

sized that the antibodies furthest from the conjugate pad

would suffer the most from loss of reagents, and thus placed

the least sensitive detection antibodies closest to the conju-

gate pad (Fig. S2A†). Specificity testing indicated three non-

specific reactions (Fig. S2B†). To investigate the lack of

242 |Lab Chip,2016,16,242–245 This journal is © The Royal Society of Chemistry 2016

Inflammation and Healing Research Cluster, Genecology Research Center, School

of Science and Engineering, University of the Sunshine Coast, Locked Bag 4,

Maroochydore DC, Queensland 4558, Australia. E-mail: jmacdon1@usc.edu.au

Division of Experimental Therapeutics, Department of Medicine, Columbia

University, New York, NY 10032, USA. E-mail: jm2236@columbia.edu

†Electronic supplementary information (ESI) available. See DOI: 10.1039/

c5lc01323b

Lab Chip,2016,16,242–245 | 243This journal is © The Royal Society of Chemistry 2016

binding for BODIPY®FL and dansyl antigen–antibody pairs,

we reduced the LFD to a 7-dot array containing six detection

antibodies (chosen due to their superior specificity; see Fig.

S2†), with a seventh detection antibody of either anti-

BodipyFL, anti-dansyl, or anti-Cy5 detection antibody (Fig. 2A;

anti-Cy5 was included as a comparative control). These

were tested with a single dual-labeled analyte containing

either 3′BODIPY®FL, dansyl, or Cy®5 antigens, or with

an analyte reaction mix containing the corresponding seven

antigens. Interestingly, the BODIPY®FL antigen still did not

produce any observable test dots, whereas the dansyl test dots

appeared and showed specificity in the seven antigen-

antibody pairs system (Fig. 2). The lack of BODIPY®FL binding

(Fig. 2 & S2†) is most likely due to the larger surface area

ablating detection, since BODIPY®FL was the least sensitive

in the single-plex assays (Fig. 1). However, the lack of dansyl

binding in the 3 ×4 array but successful binding in the 7-dot

array was consistent in multiple tests. This suggests the pres-

ence of other detection antibodies precluded dansyl binding,

despite it being placed in the first row of the 3 ×4 array. The

7-dot array that incorporated dansyl (Fig. 2), demonstrated

significant relative higher dot intensity (p<0.05) compared

to the Cy®5 (Fig. 1 and 2), and was used for all subsequent

demonstrations. These results support the notion that

specificity is a hindrance in expansion into multiplexing, but

with judicious design and repeated testing, compatible com-

binations can be identified that do not cross-react but

instead reproducibly give selective responses.

On a linear scale, our 7-dot LFD array is an improvement

on the current maximum multiplex detection that employs

antigen–antibody reactions, since only penta-plex LFD has

been previously reported.

However, stacking the test lines

(or dots) along the flow path of the lateral flow device does

not provide an intuitive result output.

Inspired by the

electronic information display systems used in digital

watches and elevators, we considered development of a

proof-of-concept 7-segment LFD display. We devised a detec-

tion system to represent each segment of a 7-segment display

with one detection antibody (from the seven) (Fig. 3A and S3A†).

Fig. 2 Hepta-plex lateral flow detection results for combinations of

seven antigen–antibody pairs. A: Positioning of detection antibodies

deposited upon lateral flow strips. B: Hepta-plex lateral flow detection of

1μM dual-labeled analyte alone (containing either 3′BODIPY®FL, Cy®5,

or dansyl), or combinations of dual-labeled analyte (1 μM) containing 3′

biotin, Cascade Blue®, digoxigenin, dintrophenyl, tetramethylrhodamine

(TAMRA), and Texas Red®and either BODIPY®FL, Cy®5 or dansyl, as

indicated. Assay was repeated three times and a representative photo-

graph is shown. Experimental details are described in the ESI.†

Fig. 3 7-Segment display of numbers on lateral flow strips. A:

Positioning of the detection antibodies to form the 7-segments of the

display. B: Addition of labeled analyte signature mixtures (1 μM each)

resulted in the successful appearance of numbers (0 to 9). The assay

was performed three times with similar results; a photograph of one

test is shown. Experimental details are described in the ESI.†

Fig. 1 (A) Design of single-plex LFDs: a dual-labeled analyte (RVFV

DNA) was sandwiched between capture and detection antibodies due

to antigen–antibody binding; AuNPs conjugated to the capture anti-

body (mouse anti-fluorescein antibody) enabled visualization of bind-

ing through the appearance of a red color at the test dot; rabbit anti-

mouse antibody, which can directly bind the capture antibody (mouse

anti-fluorescein), was deposited in parallel as a control. (B) Determina-

tion of detection limits for each single-plex assay: detection antibodies

corresponding to the 3′antigen of dual-labeled RVFV were deposited

at the test dot; resultant assay color intensity (average and standard

deviation of 4 individual tests) was quantified using ImageJ software

and plotted against DNA concentration; a cut-off defined as three

standard deviations above the average was used to determine the limit

of detection for each assay. Experimental details are described in the

ESI.†

Lab on a Chip Communication

244 |Lab Chip,2016,16,242–245 This journal is © The Royal Society of Chemistry 2016

Initially we used duplicate test dots to represent each seg-

ment, however, this resulted in position effects where the

morphology of the second test dot was influenced by the first

test dot in the vertical segments (Fig. S3†). We thus reduced

the vertical segments to only incorporate one test dot

(Fig. 3A). By addition of appropriate label mixtures, we con-

sistently demonstrated clear numbers (0–9) on the 7-segment

LFD display (Fig. 3B).

Our successful demonstration of the numbers 0–9 using

seven antigen–antibody pairs on a LFD is the first digital-like

display of numbers on a paper-based biosensor. It operates

as a single-use static output that employs a pre-defined

molecular encoding strategy for information transfer. The

work is a significant advance on our previous molecular

7-segment display, based on molecular logic gates, which

required multiple additions of samples to each segment.

Here the solid-phase LFD enables a single addition of sample

to create a display, made possible through the novel combi-

nation of both molecular and binary encoding. This is nota-

bly different from other LFD computational strategies, which

showcase embedded molecular logic gates.

9,10

Our method

also advances previous multiplex LFDs, which use multiple

lines,

7,11

bi-directional,

12,13

parallel,

14,15

and multi-directional

systems.

16,17

Notably, intuitive read-outs were previously pro-

posed by Shen et al., where blood types on a paper-based bio-

sensor were displayed using letters deposited in different sec-

tions of the biosensor (not overlayed).

18,19

However,

expansion of these multiplex LFDs is hampered by either

space limitations within the original device dimension or the

requirement to consume more reagents as the device size

increases. In contrast, our novel combination of both binary

and molecular encoding demonstrates improved multiplexing

efficiency while minimizing excess reagent requirements.

The most compact LFD system to date is the microarray

LFD,

20,21

which similarly minimizes both reagent consump-

tion and device dimension. The largest demonstration to

date is detection of 384 recombinant protein antigens for

analysis of human protein atlas antibody cross-reactivities.

Here we provide further compaction by applying binary

encoding. Our generic antigen–antibody method could theo-

retically detect 127 (2

−1) discrete analytes using only 7

antigen–antibody combinations. Noticeably, our system would

only apply to differentiation of discrete analytes: different

encoding schemes would need to be employed to differenti-

ate analyte mixtures. However, implementation of both

binary-encoded and traditional microarray LFD requires

external readers to interpret results. Our 7-segment LFD

intentionally restrict outputs to a numerical system to dem-

onstrate an intuitive read-out applicable to point-of-care diag-

nostics that does not require external reading devices.

In this study we used an artificial ideal analyte for proof-

of-concept demonstration of multiplex LFD displays. Impor-

tantly, these displays are generic and can be applied for the

detection of any candidate analyte (e.g. nucleic acids, proteins,

lipids, or small molecules) through the use of labeled binding

partners. Practically, this could be implemented via labeled

antibodies or aptamers for detection of their corresponding

antigens. In addition, we are also considering labeled

primers and probes for multiplex nucleic acid amplification,

which would additionally assist with detection of low copy

numbers. In this context, we note the advancement of iso-

thermal nucleic acid amplification technologies that offer

prospects for rapid point-of-care detection.

22,23

Determina-

tion of analyte concentration could be performed using

electronic reading devices (that assess intensity within a

defined test area regardless of dot morphology) and by pre-

training with known concentrations of analyte. Notably,

implementation of pattern displays requires the use of mix-

tures of labeled binding partners, such that an analyte must

combine with a unique subset of labels. This means that

multiple individual sub-reactions must work synchronously,

which ultimately increases confidence that can be drawn

from a successful display.

Conclusions

Our novel 7-segment LFD enables compaction of

multiplexing by borrowing from computational science and

employing a binary encoding scheme that moves beyond

space limitations. The easy-to-interpret results via a 7-seg-

ment display format are highly relevant to point-of-care appli-

cations. These displays are generic and can be applied for

multiplex detection of any candidates (e.g. nucleic acids, pro-

tein, lipid, or small molecules) if capture reagents incorpo-

rate our specific recognition labels. This is particularly ame-

nable for nucleic acid lateral flow detection, if upstream

nucleic acid amplification uses primers and probes that

incorporate the recognition labels.

Further improvements

could focus on employing advanced deposition techniques to

improve the performance and presentation of the outputs.

Acknowledgements

We thank John Bartlett, David McMillan, and Fabrice

Rossignol for support and advice, and Richard Burns for care-

ful reading of this manuscript. This work was funded by the

Queensland Government, Department of Science, Information

Technology, Innovation and the Arts (DSITIA, Australia), and

an internal HDR grant (University of the Sunshine Coast,

Australia).

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Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

Paper-Based Diagnostic Devices for Infectious Diseases

Chapter

Full-text available

Jun 2023

Arun Sharma

Paper-based biosensor technology offers an incredible prospective for quick medical diagnosis and which is one of the best point-of-care (POC) biosensors. The main advantage of paper-based technology is that it is user-friendly, low-cost, simply and readily available (Hu et al 2017, Böhm et al 2018, Choi et al 2019). Many challenges are associated with these benefits in order to develop and commercialize it. Large scale manufacturing limitations and low performance are the main challenges connected with commercialization of paper-based biosensors (PBBs). PBBs propose an economic foundation for rapid and trouble-free recognition of a biomarker in quantifiable diagnostics and supplementary areas such as foodstuffs and ecological scrutinization. In view of the fact that paper is a multipurpose substrate that facilitates reflexive solutions currently and is biocompatible, it is appropriate to exploit it as a biochemical sensing platform. In this chapter we will discuss the state-of-the-art of paper-based technology and challenges in its commercialization and also enlighten the existing inclination of PBBs in stipulations of invention and recognition practices in medical diagnostics. Key features and schemes to boost performance and amplify commercialization are projected in this chapter.

Paper-based multiplex biosensors for inexpensive healthcare diagnostics: a comprehensive review

Article

Full-text available

May 2023
BIOMED MICRODEVICES

Multiplex detection is a smart and an emerging approach in point-of-care testing as it reduces analysis time and testing cost by detecting multiple analytes or biomarkers simultaneously which are crucial for disease detection at an early stage. Application of inexpensive substrate such as paper has immense potential and matter of research interest in the area of point of care testing for multiplexed analysis as it possesses several unique advantages. This study presents the use of paper, strategies adopted to refine the design created on paper and lateral flow strips to enhance the signal, increase the sensitivity and specificity of multiplexed biosensors. An overview of different multiplexed detection studies performed using biological samples has also been reviewed along with the challenges and advantages offered by multiplexed analysis.

Dual-Mode Logic Gate for Intelligent and Portable Detection of MicroRNA Based on Gas Pressure and Lateral Flow Assay

Article

Mar 2023
ANAL CHEM

Molecular logic gate provides an intelligent option for simultaneous detection of biomarkers. Herein, a dual-mode DNA logic gate was proposed to portably and intelligently detect multiple microRNAs (miRNAs) by gas pressure biosensing and lateral flow assay (LFA). A platinum-coated gold nanoparticle (Au@PtNP) with catalase-like activity was used as a signal reporter to achieve a dual-signal readout. MiRNAs as the input initiated the cyclic strand displacement reaction (SDR) to enrich a large amount of Au@PtNPs. Thus, miRNA can be visually detected by a lateral flow strip (LFS) using the grayish-brown color of Au@PtNPs as output 1. Furthermore, Au@PtNP-catalyzed decomposition of H2O2 resulted in gas pressure as output 2, which was measured by a digital and handheld gas pressure meter. As a consequence, microRNA 21 (miR-21) was sensitively and reliably detected with the limit of detection (LOD) of 7.2 pM. The selectivity and real sample analysis were both satisfactory. Significantly, two-input and three-input AND logic gates were successfully developed to realize multiple detection of two miRNAs and three miRNAs, which provide a promising way for intelligent multi-input analysis. Predictably, with the advantages of portability, simplicity, and affordability, the dual-mode logic gate based on gas pressure biosensing and LFA offers a new perspective on the field of intelligent and portable biosensing and bioanalysis.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes

Article

Full-text available

Dec 2022

Characterization of wax valving and μPIV analysis of microscale flow in paper-fluidic devices for improved modeling and design

Article

Jun 2022

Paper-fluidic devices are a popular platform for point-of-care diagnostics due to their low cost, ease of use, and equipment-free detection of target molecules. They are limited, however, by their lack of sensitivity and inability to incorporate more complex processes, such as nucleic acid amplification or enzymatic signal enhancement. To address these limitations, various valves have previously been implemented in paper-fluidic devices to control fluid obstruction and release. However, incorporation of valves into new devices is a highly iterative, time-intensive process due to limited experimental data describing the microscale flow that drives the biophysical reactions in the assay. In this paper, we tested and modeled different geometries of thermally actuated valves to investigate how they can be more easily implemented in an LFIA with precise control of actuation time, flow rate, and flow pattern. We demonstrate that bulk flow measurements alone cannot estimate the highly variable microscale properties and effects on LFIA signal development. To further quantify the microfluidic properties of paper-fluidic devices, micro-particle image velocimetry was used to quantify fluorescent nanoparticle flow through the membranes and demonstrated divergent properties from bulk flow that may explain additional variability in LFIA signal generation. Altogether, we demonstrate that a more robust characterization of paper-fluidic devices can permit fine-tuning of parameters for precise automation of multi-step assays and inform analytical models for more efficient design.

Inkjet printing for biosensor fabrication: Combining chemistry and technology for advanced manufacturing

Article

Full-text available

Apr 2015
LAB CHIP

Inkjet printing is emerging at the forefront of biosensor fabrication technologies. Parallel advances in both ink chemistry and printers have led to a biosensor manufacturing approach that is simple, rapid, flexible, high resolution, low cost, efficient for mass production, and extends the capabilities of devices beyond other manufacturing technologies. Here we review for the first time the factors behind successful inkjet biosensor fabrication, including printers, inks, patterning methods, and matrix types. We discuss technical considerations that are important when moving beyond theoretical knowledge to practical implementation. We also highlight significant advances in biosensor functionality that have been realised through inkjet printing. Finally, we consider future possibilities for biosensors enabled by this novel combination of chemistry and technology.

Simultaneous Colorimetric Detection of Improvised Explosive Compounds using Microfluidic Paper-Based Analytical Devices

Article

Full-text available

Jul 2014
Anal Methods

In this paper the development of microfluidic paper-based analytical devices (µPADs) is described for the rapid, on-site detection of improvised explosives. Five lane µPADs were designed and printed using wax ink on chromatography paper to create hydrophobic channels. Each channel contains colorimetric reagents capable of reacting with one or more explosive compounds resulting in a specific colorimetric reaction. Two devices were prepared, each capable of performing five simultaneous analyses on a single µPAD. The first µPAD was developed to detect inorganic explosives such as black powder, flash powder, and ammonium nitrate. It detects nitrates, nitrites, chlorates, and perchlorate oxidizers, as well as ammonium. The second µPAD was developed to detect military explosives such as TNT and RDX along with other high explosives like urea nitrate. It also detects organic peroxides such as TATP and hydrogen peroxide. All experiments were performed by dissolving the explosives in deionized water or 50:50 acetone/ H2O as transport solvents with a detection time of around 5 minutes. Detection limits ranged from 0.39 – 19.8 µg of explosive compound. These two customized µPAD devices permit the on-site forensic testing of unknown explosives, thereby supplying law enforcement and military personnel with a resource for fast, easy detection of military, commercial, and homemade explosive components at low cost.

Multiplexed paper test strip for quantitative bacterial detection

Article

Full-text available

Apr 2012
ANAL BIOANAL CHEM

Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (β-galactosidase (B-GAL) or β-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-β-D: -glucuronide sodium salt (XG), chlorophenol red β-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

Paper-Based Blood Typing Device That Reports Patient's Blood Type "in Writing"

Article

Full-text available

May 2012
ANGEW CHEM INT EDIT

A response in writting: A low-cost bioactive paper device is designed to perform ABO and RhD blood typing tests, and the paper reports the results in writing. This idea was inspired by the vision of the British author, J. K. Rowling, through her novel "Harry Potter and the Chamber of Secrets" in which a piece of paper could be interrogated for information and unambiguous answers were received from the paper in writing.

Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics

Article

Oct 2015

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

A universal lateral flow biosensor for proteins and DNAs based on the conformational change of hairpin oligonucleotide and its use for logic gate operations

Article

Jun 2014
BIOSENS BIOELECTRON

Paper-based device for rapid typing of secondary human blood groups

Article

Nov 2013
ANAL BIOANAL CHEM

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.

The Dynamics of Capillary How

Article

Jan 1921

Edward W. Washburn

Penetration of Liquids into Cylindrical Capillaries.—The rate of penetration into a small capillary of radius r is shown to be: dldt=P(r2+4εr)8ηl, where P is the driving pressure, ε the coefficient of slip and η the viscosity. By integrating this expression, the distance penetrated by a liquid flowing under capillary pressure alone into a horizontal capillary or one with small internal surface is found to be the square root of (γrt·cosθ2η), where γ is the surface tension and θ the angle of contact. The quantity (γcosθ2η) is called the coefficient of penetrance or the penetrativity of the liquid.

Computational Lateral Flow Biosensor for Proteins and Small Molecules: A New Class of Strip Logic Gates

Article

Jul 2012
ANAL CHEM

The first example of strip logic gates ("OR" and "AND" functions) for proteins and small molecules has been constructed on the basis of target-induced self-assembly of split aptamer fragments. Using thrombin and ATP as inputs, the corresponding split/integrated aptamers as molecular recognition elements, and gold nanoparticles as a tracer, the output signals can be directly visualized by observing the red bands on the test zones of the strips. The assay is simple, easy to perform, and cost-effective, allowing portable analysis at ambient temperature. The strip logic system is resistant to nonspecific interfering agents and can operate effectively even in human serum samples. Such logic strips hold great promise for application in intelligent point-of-care and in-field diagnostics.

Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus

Article

Jun 2012

Detection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. To develop an isothermal 'recombinase polymerase amplification (RPA)' assay on an ESEquant tubescanner device. RPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. The one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log(10) step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.