Point-of-Care Multiplexed Assays of Nucleic Acids Using

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Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-based Loop-Mediated Isothermal Amplification Yi Zhang,†,‡,⊥ Lu Zhang,†,⊥ Jiashu Sun,*,† Yulei Liu,§ Xingjie Ma,∥ Shangjin Cui,∥ Liying Ma,§ Jianzhong Jeff Xi,‡ and Xingyu Jiang*,† †

Beijing Engineering Research Center for BioNanotechnology & Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China ‡ Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China § State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention (NCAIDS), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention (China-CDC), Beijing 102206, China ∥ State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of CAAS, Harbin 150001, China S Supporting Information *

ABSTRACT: This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings.

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its geometry,12,14−16 such works increased the complexity and the cost of the fabrication of devices. We therefore considered a straightforward microreactor composed of other materials specifically for the contamination-free and miniaturized LAMP reaction. Multiplexed detection of nucleic acids in a single inexpensive and easy-to-operate device is still in urgent need for practical POC diagnostics in resource-poor settings, such as areas of limited healthcare infrastructure, or private households. We proposed two on-chip LAMP schemes to carry out simultaneous amplification of multiple targets, previously.8,9 These prototypes showed the promise of simplicity, but the preparation of the devices still relies on advanced instruments. Some chips were just the scaled-down versions of PCR strip tubes, and the reagent consumption was not reduced dramatically so far, or even larger than the primitive Eppendorf tubes.17 In the past decade, various droplet-based techniques have been developed for the amplification of nucleic acids in arrays.18−21 However, these techniques involve many complex fluidic controls as well as additional external supplies outside

ow-cost and miniaturized point-of-care (POC) nucleic acid detection has been developed extensively in recent years. Powerful and straightforward, loop-mediated isothermal amplification (LAMP) is an attractive method for detecting nucleic acids because of its isothermal process and high specificity.1 The combination of the isothermal amplification and microfluidics has shown great potential for developing POC devices.2,3 The performance of LAMP methodology was verified in many systems in the past decade. LAMP may be, however, more sensitive to minute contamination than PCR, which severely hinders its application.4,5 Our previous work showed polydimethylsiloxane (PDMS)-based microreactors to carry out miniaturized and multiplexed LAMP reactions.6−9 Intrinsically, PDMS is a porous and gas-permeable material. The porosity or the gas/vapor permeability has been widely utilized for many biological/chemical studies,10,11 but on the other hand, these advantages may become the major obstacle for nucleic acid detection due to the bubble formation or vapor evaporation in the PDMS-based microreactors.12 Bubbles can generate during the process of loading and heating the solution because of the hydrophobicity of PDMS or the irregular microgeometry.13 The inflation of bubbles may further burst the sealing of inlet/outlet during the curing stage of the PDMS, thus resulting in contamination.8,9 Although some works modified the original PDMS to improve its impermeability or © 2014 American Chemical Society

Received: April 19, 2014 Accepted: June 17, 2014 Published: June 17, 2014 7057

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Figure 1. Schematics of cLAMP device. Two types of sample introduction (a and b): (a) capillary force and positive pressure-driven introduction. We contact the top of the tip with the end of the microcapillary and depress the plunger of the pipet to the second (not the first) stop, which results in a liquid drop followed by a section of air. (b) Introduction by displacement. We prefill the microcapillary with pure water and dispense a drop of liquid to the top end of the microcapillary while contacting the bottom end of the microcapillary with an absorbent paper (KimWipes), which results in a fluid (liquid and gas) flow that introduces the sample in segments of liquids within the microcapillary. (c) Schematic representation of a typical result of cLAMP. The ends of microcapillary are sealed by a set of segments of pure water (liquid) and air (gas), and epoxy glue (solid), which results in a triple insurance. On the basis of this basic construction, the different LAMP premixes in the middle of the microcapillary are separated by segments of water.

plasmid by Sangon Biotech (Shanghai). Plasmids containing the target nucleic acid fragments were used as the standard, and the detailed sequences of the fragments are in the Supporting Information, Table S1. The sets of primers corresponding to each plasmid are synthesized by Sangon Biotech, and the detailed sequences of the primers are in the Supporting Information, Table S2. The viral RNA of HIV was extracted from drug-resistant human blood plasma (numbered by China CDC as DRC 124 J, DRC 257 J, and DRC 305 J, respectively) using PureLink Viral RNA/DNA Kits (#12280-050, Invitrogen), according to the manufacturer’s protocol. DRC 124 J and DRC 305 J resist the treatment with a cocktail of “AZT+NVP+3TC”, and DRC 257 J resists the treatment with a cocktail of “3TC+TDF+LPV”. Two sets of reverse transcription LAMP (RT-LAMP) primers were reported previously,28 each recognizing a target sequence located within either the HIV-1 p24 or protease gene. The detailed sequences of primers are in the Supporting Information, Table S2. We employed the two-component epoxy glue to seal the ends of the microcapillary (inner diameter (ID) of 500 μm, outer diameter (OD) of 1000 μm, and 100 mm in length) within minutes. Many manufactures can produce inexpensive epoxy glues with quick-curing properties (e.g., 5 min Epoxy Gel by Devcon, 5 min Z-POXY by ZAP, Scotch-Weld Epoxy Adhesive DP-105 by 3M). Besides the glass microcapillary, for comparison purposes, we also tested microcapillaries made of other materials. Flexible

the reaction chips. Most control systems require at least a syringe pump, which may not be applicable to POC settings. To avoid complex operation and decrease the heat-induced liquid evaporation in microscale during the nucleic acid amplification, some open systems based on capillary channels have been proposed.13,22−24 However, sealing is still the predominant and convenient strategy for a contamination-free system. The common sealing methods have some challenging issues such as high complexity or ineffectiveness,7 thus a simple but effective sealing method is still needed. To address these challenges, capillary-based nucleic acid detection methods have been developed with minimal fabrication complexity and easy parallelization.25−27 These sensing schemes still involve the use of an external pumping system and/or heating block. In this work, we demonstrate a straightforward and multiplexed detection of nucleic acids by integrating LAMP into a microcapillary system (cLAMP) and using segments of water droplets to isolate each amplification reaction as well as preventing contamination. This cLAMP system introduces sample/reagents by capillary action, uses pocket warmers for gene amplification, and applies a hand-held flashlight for readout, without relying on external power supply and equipment.

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EXPERIMENTAL SECTION Materials. Conserved nucleic acid fragments of pandemic influenza A H1N1 virus (pandemic H1N1) and influenza A virus (Flu A) were screened and cloned into the pUC57 7058

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1.0 M betaine, 0.32 U/μL Bst polymerase, and a small amount of sample. Amplification was performed at 65 °C in an oven, or heated by the pocket warmer (Figure 2), for 1 h. The final result was directly determined by the naked eye based on the appearance of the green fluorescence induced by calcein.

fused silica capillaries with different inner/outer diameters coated with standard polyimide or high temperature-resistant polyimide coating were from Polymicro Technologies (#TSP530660, #TSP530700, #TSG530660). Acrylic (PMMA) capillaries with 500/750 μm (ID/OD) or 500/ 1000 μm (ID/OD) and polytetrafluoroethylene (PTFE, also known as Teflon) microcapillaries with 559/1067 μm (ID/ OD) were from Paradigm Optics (#CT500-750-5, #CT5001000-5, #CTPT559-1067-5). Thinner-walled PTFE microcapillaries with 610/914 μm (ID/OD) were from NewAge Industries (#3300423). Clear C-FLEX microcapillaries (made out of modified styrene−ethylene−butylene block copolymer) with 510/2100 μm (ID/OD) were from Cole-Parmer (#EW06422-00). The common features of these microcapillaries include flexibility, transparency (for the naked-eye), and the ability to withstand temperatures >90 °C. We have to practically assess the UV-transparency of each material in this study, because there are not always sufficient data from the manufacturers or other publicly available information. A hand-held UV-flashlight (ZY-S12, Small Sun) was purchased from an online store (www.taobao.com), and used to excite the fluorescence produced by LAMP. It is powered by a 3000 mAh rechargeable Li-ion battery (UltraFire) and a single LED emits 365 nm UV-light with a power of 3 W. We wear UV-protective glasses for observing the fluorescence, and the fluorescence images were captured by a digital camera (DMCFX520, Panasonic). In this study, we used calcein (#340-00433, DOJINDO) as the fluorescence indicator. Bst DNA polymerase with attached ThermoPol buffer (#M0275S), and AMV reverse transcriptase (#M0277S) were from New England Biolabs. Betaine (#B0300) solution and manganese(II) chloride (#203734) were from Sigma-Aldrich. Other chemicals were from Aladdin. For an alternative heating strategy, a disposable pocket warmer (www.taobao.com) was used to heat the microcapillaries.29 Device Assembly. To prevent contamination, we carried out cLAMP in an enclosed glass microcapillary. Among the commonly used materials for fabricating capillaries, glass shows significant advantages in medical applications because of its excellent biocompatibility, favorable optical transparency, and stable physicochemical property. By using capillary forces,30,31 all reactants were initially added before the amplification. Pumping-based liquid injection into a capillary channel for multiplexed nucleic acids amplification was extensively studied in the past decade,32,33 and in this study, the liquid of interest was introduced into the microcapillary by capillary force without the need of large external pumping equipment. This process was very fast (within seconds) and convenient. We sealed the ends of the microcapillary by fast-curing epoxy glue (Figure 1c), after the sequential introduction of the liquids (Figure 1a,b, and Movie S1 in the Supporting Information). Finally, the ends of the microcapillary were sealed by pure water (liquid), the air (gas), and the epoxy glue (solid), which form a triple insurance against leakage of any materials that might cause contamination. LAMP Procedure. (1). Detection of the Plasmids Containing the Conserved Fragment of Pandemic H1N1 or Flu A. LAMP was carried out in the system containing 1× ThermoPol Buffer (containing 2.0 mM MgSO4), totally 8.0 mM MgSO4, 0.5 mM MnCl2, 25.0 μM calcein, 0.4 mM deoxyribonucleotide triphosphates (dNTPs), 0.2 μM each of the outer primer (F3 and B3), 2.0 μM each of inner primer (FIP and BIP), 0.8 μM each of the loop primer (LF and LB),

Figure 2. Electricity-free heating for cLAMP by a portable pocket warmer. (a) Photograph of the pocket warmer. (b) Schematic assembly. The plastic film could prevent the outer surface of microcapillaries from being defiled by the residual of the adhesive. (c) Detection of two amplified target plasmids inside a capillary heated by pocket warmers.

(2). Detection of the Viral RNA of HIV. We carried out RTLAMP according to previous reports with minor revisions.28 This system contains 1× ThermoPol Buffer, 10.0 mM MgSO4, 0.5 mM MnCl2, 25.0 μM calcein, 1.4 mM dNTPs, 0.2 μM each of the outer primer (F3 and B3), 1.6 μM each of inner primer (FIP and BIP), 0.8 μM each of the loop primer (LF and LB), 0.4 M betaine, 0.32 U/μL Bst polymerase, 0.025 U/μL AMV reverse transcriptase, and a small amount of extracted RNA. Amplification was performed at 65 °C for 45 min. The final result was directly determined by the naked eye based on the appearance of the green fluorescence induced by calcein.

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RESULTS AND DISCUSSIONS We carried out multiplexed LAMP reactions in a segment format within a glass microcapillary. The liquid samples were introduced into the capillary with a smooth internal wall by capillary force (Figure 1, Movie S1 in the Supporting Information). This operation was rapid and convenient as compared to many microfluidic devices. Because of the smooth inner surface, we could completely repress the formation of bubbles during the sample introduction and the subsequent heating process.12,13 To avoid the cross-talk between two reaction zones, we separated them by spacing two segments of pure water (Figure 3a−c). These water segments would also help reduce the evaporative loss/contamination of liquid samples during thermal reactions, by replenishing water vapor and decreasing the vapor concentration gradient inside the capillary.23 To prevent any possible evaporation/leakage of samples, we sealed 7059

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premixed into the reaction solution. Calcein is a fluorescent indicator for alkaline metal ions (such as Mg2+, Ca2+) by forming complexes. This fluorescence could, however, be quenched by some other ions (such as Mn2+, Co2+, Ni2+, Cu2+, Fe3+). In specific case of LAMP, both competitive Mg2+ (with relatively higher concentration) and Mn2+ (with relatively lower concentration) ions are preadded into the solution, and the fluorescence of calcein is initially quenched by Mn2+ before reaction. As amplification proceeds, Mn2+ forms an insoluble salt with the pyrophosphate byproduct released from dNTPs. Calcein is thus deprived of Mn2+, and instead combines with residual Mg2+, producing a detectable fluorescence.34 Others dyes such as SYBR green I35 or propidium iodide36 are typically added to the solution after the completion of the reaction. This operation of adding these dyes would increase the steps of operation and introduce risks of contamination. We used a high-power hand-held UV-flashlight to excite the fluorescence of calcein. Compared with other commonly used desktop UVlight sources, the single LED design dramatically reduced the dimension of the system without loss of functionality. We also tested other portable UV-flashlight emitting longer wavelength such as 405 nm, or with low-power LED,37 and the results proved that high-power 365 nm wavelength is the best for exciting bright fluorescence (Supporting Information, Figure S1). A major advantage of the microcapillary is the ease of parallelization, which allows multiplexed assays or assessment of the sensitivity of the assay via serial dilution. The level of parallelization was related to the luminous area of the light source. In our case, by using a hand-held UV-flashlight, we could parallelize 8−10 capillaries in a single experiment. We prepared seven samples with different DNA concentrations from 2 to 2 × 106 copies by serial dilutions of standard plasmid (initial concentration of 1.1 × 1010 copies/μL) before amplification. As all diluted DNA samples generated fluorescent signals by cLAMP in a parallel format, we concluded that the minimum number of copies that could be amplified was 2 (Figure 4). The sensitivity (minimum detectable quantity) of

Figure 3. Multiplexed cLAMP carried out in a variety of forms through partition of microcapillaries. (a) Simultaneous detection of two influenza A subtypes, influenza A virus and the 2009 pandemic influenza A H1N1 virus. Panels b and c show that there is no cross-talk between test samples by spacing two segments of water. (d) Samples of random volumes from 100 nL to 2.5 μL could be detected by cLAMP.

the two ends of the capillary with epoxy glue, which was commonly adopted for rapidly bonding glass, plastics, or many other materials at room temperature. The maximum tolerable temperature of epoxy glue is up to ∼90 °C, which is much higher than the typical working temperature of around 65 °C required by LAMP. After multiplexed cLAMP was carried out through partition of the capillary, there was no observable evaporation, indicated by the imperceptible contraction of the reaction solution (Figure 3a−c). The white dots in Figure 3 were caused by the light reflection at boundary of the liquid droplet and the air segment, which did not interfere with our assay. Moreover, we introduced six samples of random volumes from 100 nL to 2.5 μL into the capillary, which were separated from each other by two water segments. All samples could be detected after amplification, indicating that the cLAMP was insensitive to the volume of samples or spacing (Figure 3d). These above features of cLAMP ensured a straightforward, robust, and multiplexed detection of nucleic acids, while preventing evaporation and cross-talk of samples, reducing the complexity of the system, and facilitating the fluidic control. All experiments in the present work were carried out for at least three times, and showed a consistent result. Empirically, the primer design is critical to the success and stability of LAMP, which, in this study, was optimized for many rounds, ensuring the reproducibility of reaction. The naked eye-based readout of cLAMP was realized by using calcein as the fluorescence indicator, which could be

Figure 4. Sensitivity test of cLAMP using standard plasmid with different concentrations (the 2009 pandemic influenza A H1N1 virus) in parallel microcapillaries. The results showed that cLAMP could amplify and detect as low as 2 copies of DNA. No template represents zero copy of plasmid.

conventional LAMP in a tube is around 10 copies, and we successfully detected as low as 2 copies of standard plasmid in our experimental settings. The reaction time of cLAMP was dependent on the initial concentration of DNA. For 2 × 106 copies of DNA, the amplification time was around 15 min for obtaining a visible readout. We further carried out amplification in the combined format, i.e., segment assay in parallelized format, to detect viral nucleic acids from multiple human plasma samples (Figure 5). Each microcapillary corresponds to 7060

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where the brittle glass is a disadvantage, for example, when rough handling is unavoidable. To enable this assay in real POC scenarios, we carried out the cLAMP as above by using an electricity-free heater. We validated the feasibility of heating by the disposable pocket warmer (Figure 2). The cost per piece is less than 0.16 US dollars. The warmer could reach a maximal temperature of 68 °C and maintain the average temperature at 53 °C during a 12 h period. We sandwiched microcapillaries between two pieces of pocket warmers during the amplification. It should be noted that the adhesive on the surface of the warmer emitted strong blue fluorescence under UV irradiation. Thus, we need to insert two pieces of thin plastic films between microcapillaries and two pocket warmers to prevent the outer surface of the microcapillaries from being contaminated by the residual of the adhesive (Figure 2b). After incubation for 1 h by pocket warmers, the amplified target plasmids could be detected with the naked eye, and the results agreed well with those cLAMP heated inside the oven (Figure 2c). We compared our cLAMP with other reported variations of LAMP system and standard PCR in Table 1. To the best of our

Figure 5. Multiplexed detection of HIV virus. We detected two target sequences (p24 gene and protease gene) of HIV from three drugresistant control (DRC) samples prepared from the blood of different individuals. Each sample (numbered as DRC 124 J, DRC 257 J, and DRC 305 J) corresponds to one microcapillary, and each microcapillary detects these two sequences.

one sample, and we detected two target sequences (p24 gene and protease gene) of HIV from three DRC (drug-resistant control) samples to confirm the HIV infection. These results revealed the flexibility of the system beyond other strategies for the miniaturized nucleic acid amplification. To show that this assay is compatible with capillaries made of different materials, we performed cLAMP by using a variety of flexible capillaries. The coating of polyimide onto the outer surface of glass/fused silica microcapillary would make it flexible and less vulnerable (Supporting Information, Figure S2a−c). Other flexible microcapillaries made by PTFE, CFLEX, or PMMA were also used as the reactors for cLAMP (Supporting Information, Figure S2d−h). After amplification inside flexible capillaries, we compared the optical readout of glass-based microcapillaries and nonglass-based ones. As the coated glass capillaries were opaque, we opened detection windows by carefully removing the polyimide layer before irradiating the samples with UV light. The amplified target samples (Flu A) inside flexible glass capillaries showed green fluorescent bands while negative controls remained nonfluorescent, which could be distinguished by the naked eye (Supporting Information, Figure S2a−c). In comparison, flexible capillaries made by PTFE and C-FLEX were translucent and weakly fluorescent under UV light (Supporting Information, Figure S2d−f), which interfered the naked-eye-based observation of fluorescence arising from the LAMP reaction. PMMA capillaries, however, were transparent with a low background fluorescence, and thus suited for performing cLAMP. These results indicated an indistinguishable difference of fluorescent intensity between the PMMA-based microcapillaries and the glass-based microcapillaries (Supporting Information, Figure S2g−h). The only shortcoming of PMMA capillaries was the relatively high rate of moisture absorption, which might result in undesired water loss as well as the loss of reaction volume over the long-term (Supporting Information, Figure S3). However, we did not observe appreciable negative influences during ∼1 h for our LAMP reactions. Moreover, we carried out the same LAMP reactions in microcapillaries with different wall thicknesses, and did not find perceptible differences among these different conditions under our experimental settings (Supporting Information, Figure S2a,b, Figure S2d,e, Figure S2g,h). Being applicable in other types of capillaries allows this assay to be readily applicable in scenarios

Table 1. Comparison between cLAMP and Other Amplification-based Methods properties function integration total volume (μL) total time (min)a total costb electricity readout throughput manipulation

cLAMP

commercial LAMP

other on-chip LAMP

commercial PCR

high

low

medium

low

10−1−1

≥25

≥10

≥20

20−90

90−240

120−240

120−240

low optional eye medium

medium necessary instrument low

medium necessary instrument low

simple

complicated

high optional instrument medium or low simple

complicated

The “total time” includes the time required for sample preparation, nucleic acid extraction, amplification, and final readout. bThe “total cost” refers to the cost of each run and the cost of system construction. a

knowledge, this is the smallest volume for a single run of LAMP reaction (∼200 nL). Although we prepared the functional microcapillary individually in the current proof-of-concept stage, this step could be easily scaled up. The possibility of electricity-free will be favored by on-site detection in remote areas. As we mentioned before, the cost of the cLAMP platform is extremely low, which is attractive for real applications.

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CONCLUSIONS We present a point-of-care assay for nucleic acid, based on microcapillaries for the miniaturized and multiplexed isothermal amplification of nucleic acids. Our cLAMP system overcomes the problem of contamination in conventional LAMP and achieves a high sensitivity of two copies of standard plasmid. We believe that this cLAMP can be adopted into many practices without resorting to complex instrumentation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 7061

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AUTHOR INFORMATION

Corresponding Authors

*X. Jiang. E-mail: [email protected]. *J. Sun. E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministry of Science and Technology (2011CB933201, 2012AA030308, 2012AA022703, and 2013AA032204), the National Science Foundation of China (51073045, 21025520, 2122058, 21222502, 91213305, 81361140345, and 51105086), Beijing Municipal Science & Technology Commission (Z131100002713024), and the Chinese Academy of Sciences (XDA09030305) for financial support. We are also grateful to Mr. Sha He for literature services.

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