Multiplexed instrument-free meningitis diagnosis on a polymer/paper hybrid microfluidic biochip

Abstract

Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae), and Haemophilus influenzae type b (Hib) are three most common pathogens accounting for most bacterial meningitis, a serious global infectious disease with high fatality, especially in developing nations. Because the treatment and antibiotics differ among each type, the identification of the exact bacteria causing the disease is vital. Herein, we report a polymer/paper hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) for multiplexed instrument-free diagnosis of these three major types of bacterial meningitis, with high sensitivity and specificity. Results can be visually observed by the naked eye or imaged by a smartphone camera under a portable UV light source. Without using any specialized laboratory instrument, the limits of detection of a few DNA copies per LAMP zone for N. meningitidis, S. pneumoniae and Hib were achieved within 1 hour. In addition, these three types of microorganisms spiked in artificial cerebrospinal fluid (ACSF) were directly detected simultaneously, avoiding cumbersome sample preparation procedures in conventional methods. Compared with the paper-free non-hybrid microfluidic biochip over a period of three months, the hybrid microfluidic biochip was found to have a much longer shelf life. Hence, this rapid, instrument-free and highly sensitive microfluidic approach has great potential for point-of-care (POC) diagnosis of multiple infectious diseases simultaneously, especially in resource-limited settings.

A paper/polymer hybrid CD-like microfluidic SpinChip integrated with DNA-functionalized graphene oxide nanosensors for multiplex qLAMP detection

“…7–17 Our group reported polydimethylsiloxane (PDMS)/paper hybrid microfluidic devices integrated with LAMP for rapid and sensitive infectious disease diagnosis. 18, 19 With the benefits of multiple compartments in microfluidic devices, multiplexed pathogen detection was reported by performing parallel singleplex LAMP reactions in different independent compartments of a microfluidic chip (note this is not mLAMP). 20–22 However, it usually can only qualitatively identify the occurrence of amplifications based on the byproducts from the reactions, and the reagent consumption and cost will be doubled, tripled or even more compared with mLAMP detection.…”

Microfluidic Platforms for the Detection of Disease Biomarkers

“…Its high surface to volume ratio provided by macro-porous structure improves detection limit for colorimetric method and also improves the immobilization of proteins, enzymes, biosensors, and DNA biomarkers [9]. The ability to store reagents in active form within the 3D structure for months at room temperature is also an important feature [15]. These paper-based devices can be fabricated either by construction of hydrophobic barriers or twodimensional cutting.…”

Diagnostic accuracy of the LAMP assay for Neisseria meningitidis

“…The overall incidence of meningitis has declined in recent decades due to the increasing use of meningococcal vaccines [5] , but the incidence of meningitis from bacterial strains not covered by vaccines has increased, and bacterial meningitis remains a significant public health problem. About 200,000 patients worldwide die from bacterial meningitis every year, and the mortality can be up to 60% in parts of sub-Saharan Africa and in poor and developing countries [6,7] . Patients who receive treatment have a mortality rate of about 10%, but survivors often experience serious sequelae, including limb amputation, neurological deficits, and other serious disabilities [8] .…”

“…For example, PCR and ELISA can be time-consuming, difficult to perform, and relatively expensive [10] . Identification by bacterial culture can take many days, has low sensitivity, and has a reduced detection rate for patients who were pretreated with antibiotics [6] .…”