Advances in microfluidics in combating infectious diseases

Abstract

One of the important pursuits in science and engineering research today is to develop low-cost and user-friendly technologies to improve the health of people. Over the past decade, research efforts in microfluidics have been made to develop methods that can facilitate low-cost diagnosis of infectious diseases, especially in resource-poor settings. Here, we provide an overview of the recent advances in microfluidic devices for point-of-care (POC) diagnostics for infectious diseases and emphasis is placed on malaria, sepsis and AIDS/HIV. Other infectious diseases such as SARS, tuberculosis, and dengue are also briefly discussed. These infectious diseases are chosen as they contribute the most to disability-adjusted life-years (DALYs) lost according to the World Health Organization (WHO). The current state of research in this area is evaluated and projection toward future applications and accompanying challenges are also discussed.

Keywords: Diagnostics; HIV; Infectious diseases; Malaria; Microfluidics; Point-of-care; Sepsis.

Fig. 1

Cumulative number of publications using microfluidic for the study and diagnosis of various infectious diseases (from 2005 to 2015). There were only 3 publications on SARS around 2002–2004 where there was SARS outbreak in Asian-Pacific countries. The infectious diseases that experience a steady rise in number of publications were HIV/AIDS and sepsis which affect both developing and developed nations equally. On the other hand, the number of publications in malaria and tuberculosis is stagnating despite the continued need for better diagnostic tools for resource-scarce communities. This shows that diseases prevalent in developing nations are still very much neglected by the scientific community that is concentrated in developed nations. Regrettably, many of the publications in dengue were also from groups in Latin America and South-East Asia, once again highlighting the lack of international interest in these infectious diseases. Note: Publication numbers were calculated using PubMed and ScienceDirect.

Fig. 2

A framework to evaluate the suitability of different malaria diagnostic tools in the market. The first 7 criteria are adapted from WHO (ASSURED) (Martinez et al., 2010) while the rest are adapted from Gascoyne et al. (2004). μTAS: micro-total analysis system. From the evaluation, it can be seen that microfluidic platforms can perform on par or even superior to many existing malaria diagnostics. Note: affordability is calculated without considering the fixed cost of machine.

Fig. 3

Microfluidic technologies used in malaria diagnosis. Principles for diagnosis: (A & B) deformability, (C) PCR, (D) optical and (E) magnetic. (A) Schematic illustration of working principle of a microfluidic device working with the concept of margination. iRBCs which are less deformable than healthy RBCs are displaced to the peripheral walls where they are collected. Making use of the margination phenomenon, 75% of early stage iRBCs and 90% of late stage iRBCs can be recovered, reprinted with permission from Hou et al. (2010). (B) Schematic illustration of a microfabricated deformability-based flow cytometer (i) that measures dynamic mechanical responses of RBCs. Experimental results (ii) demonstrating measured velocities of RBCs as a function of infection state for RBCs infected with late ring stage parasites at a pressure gradient of 0.24 Pa μm^− 1, reprinted with permission from Bow et al. (2011). (C) Schematic showing a microfluidic technique for purification of nucleic acids from iRBCs using isotachophoresis (ITP). LE, TE: leading, trailing electrolytes. Isotachophoresis was used to extract DNA from malaria parasites. Nucleic acid yield was maximized using counterflow that increased focusing time. A limit of detection of 0.5 parasites/nL was achieved, reprinted with permission from Marshall et al. (2011). (D) Schematic showing working mechanism of a paper based microfluidic device for automated staining of malaria parasites with an embedded microscopy window. Paper cartridge consisting of both thin (single cell layer) and thick (multiple cell layer) smears where blood is stained with acridine orange dye. The cartridge is then optically examined for iRBCs, reprinted with permission from Horning et al. (2014). (E) Schematic of a label-free microfluidic device for separation of iRBCs based on their paramagnetic characteristics. Presence of paramagnetic hemozin in iRBCs is used to separate iRBCs from healthy RBCs based on their differential lateral migration in a magnetic field gradient. Collection of ring-stage iRBCs (with low hemozin concentration) was made possible with the use of steep magnetic gradient, reprinted with permission from Nam et al. (2013).

Fig. 4

Microfluidic techniques contributing to sepsis management. Diagnosis: (A) Immunoaffinity, (B) droplet microfluidic, (C) spiral channel inertial microfluidic. Treatment: (D) straight channel inertial microfluidic, (E) margination, (F) treatment through filtration. (A) Immuno-affinity method to capture bacteria coupled with fluorescence imaging. The new technique takes just 30 min to complete compared to traditional bacteria culture that takes 2–3 days, greatly reducing diagnosis time. Reprinted with permission from Wang et al. (2012b). (B) IC 3D system where there is enrichment of bacteria and subsequent detection by fluorescence intensity. Bacteria are encapsulated in single droplets together with bacteria-specific DNAzyme. The platform is coupled with optical imaging and different species of bacteria can be differentiated by their fluorescence intensity, hence guiding therapeutic intervention. Reprinted with permission from Kang et al. (2014a). (C) Spiral microfluidic device that makes use of Dean drag forces to focus bacteria and platelets at the outer wall while RBCs and leukocytes which experience more substantial inertial lift forces focus near the inner wall. The filtration device takes 20 min to process 1 min of whole blood with 65% recovery of pathogens that could be used for downstream RNA analyses. Reprinted with permission from Hou et al. (2015). (D) Blood cleansing device to remove microorganisms from the body. The artificial biospleen mimicks the structure and role of spleen. Contaminated blood containing magnetic opsonin is passed into the microfluidic channel at high flow rate and the external magnets are used to remove pathogens bounded to the magnetic elements and discarded. Cleansed blood is then returned to the subject (rats). This process did not activate complement cascade and coagulation while reducing the amount of inflammatory cytokines in the system. Reprinted with permission from Kang et al. (2014b). (E) Microfluidic device making use of margination to remove bacteria. As less deformable RBCs transverse to the side channels, it causes the margination of bacteria and leukocytes to the peripheral outlets as well, leaving the center outlet bacteria-free. Reprinted with permission from Hou et al., (2013). (F) Massively parallel arrangement of 40 straight channels utilizing inertial microfluidic for filtration of bacteria at a flow rate of 240 mL/h. ~ 80% in pathogen depletion efficiency was achieved with two cycles of processing. Reprinted with permission from Mach and di Carlo (2010).

Fig. 5

Microfluidic technologies for HIV diagnosis. Principles of diagnosis: (A & B) Immunoaffinity, (C) electrical impedance and (D) RT-PCR. (A) Captured CD3 + CD4 + lymphocytes were stained and counted automatically by the designed software. This device allows 100 × faster speed in identifying immuno-stained lymphocytes for HIV detection. Reprinted with permission from Alyassin et al. (2009). (B) Left panel shows the sequence of steps the sample undergoes as it moves through the equipment-free microfluidic device (m-chip). Right panel illustrates the various steps of the immunoassay. The reduction of silver ions on gold nanoparticle conjugated with specific antibodies for signal amplification, facilitating readout without the use of expensive optics. Reprinted with permission from Chin et al. (2011). (C) Whole blood is introduced and RBCs are lysed, leaving a supposedly pure population of white blood cells. Total number of lymphocytes is counted followed by capture of CD4 + and CD8 + lymphocytes with microposts. Differential electrical impedance signals of the cells provide information on the degree of contamination and number of target lymphocytes. Reprinted with permission from Watkins et al. (2013). (D) RT-PCR integrated into microfluidic channel. Top panel shows the schematic and the bottom panel shows the actual device. Valves are created to isolate different steps of the process such as incubation, reaction and detection. Reprinted with permission from Lee et al. (2008).

Fig. 6

Microfluidic platforms for the diagnostic of dengue and tuberculosis. (A) Top: dengue virus-bound magnetic beads in the sample loading/mixing chamber are used to identify dengue patients' samples containing IgG and IgM. Magnetic coils are then turned on to collect the IgG/M-bound magnetic beads followed by purification and subsequent fluorescent readouts at the detection chambers loaded with antibodies. Bottom: design of micro-mixer for efficient mixing of magnetic beads and biological samples. Reprinted with permission from Lee et al. (2009). (B) Design of a stacking lateral flow paper microfluidic assay for dengue diagnostic. Saliva from patients are filtered through a paper layer made of fiber glass to remove proteinaceous substances. A detection sensitivity of 20 ng/mL of α-fetoprotein in the saliva serum is achieved. A control line is also present in the device as a positive control. Reprinted with permission from (Zhang et al., 2015). (C) A fully integrated thermoplastic microfluidic device for detection of DNA of M. tuberculosis and drug resistance with fluidic path controlled by electrically actuated solenoid. Steps such as cell lysis, DNA isolation and PCR can be performed fully on the microfluidic chip. Micro-pillars are also employed to enhance the density of adsorbed DNA for colorimetric detection. Reprinted with permission from Wang et al. (2012a).