There is a large demand for portable diagnostic equipment for point-of-care testing and for measurements at remote areas with limited health infrastructure and resource restrictions. Microfluidic devices, which are worked based on the manipulation and control of fluids physically confined to submillimeter dimensions, can be considered an ideal platform to fabricate portable diagnostic devices. Conventional microfluidic devices are fabricated by manipulating microchannels inside the substrates like glass or different plastics and the solutions flow through channels using external pumps. Considering global health, the use of environmentally friendly substrates has attracted much attention. On the other hand, to be compatible with point-of-care testing, devices without the need to power sources (such as electric pumps) are preferred. Paper and thread are two biocompatible, accessible, portable and low-cost substrates, which have widely been used for fabricating microfluidic devices. In microfluidic paper or thread-based analytical devices (µPADs and µTADs), the fluid can transport on these substrates by different driving forces such as wicking action, gravitational force, and electrophoretic force [1,2].

Microfluidic analytical devices can be applied as sensors or biosensors with a different applications in point-of-care diagnostic [3], health monitoring [4], environmental [5,6], and food safety [7,8]. Accordingly, different analytical sensing strategies based on the paper substrate have been invented [9], [10], [11]. Analytical chemists used the wicking feature of the paper to develop lateral flow assays, most specially immunoassays [12,13]. Using different paper patterning methods, one can create multi-channels microfluidics for multi-assays too. Multi-channel paper microfluidics has also been used to fabricate electronic tongues on the paper substrate, where an array of cross-reactive chemo-sensor reagents are deposited at the end of the created hydrophilic channels on the paper. Such devices produce multivariate data, which are analyzed by the chemometrics method to extract unique signature patterns for the individual analytes or systems of complex mixtures of the analytes [5,14,15].

Paper-based titration can be considered as an innovative application of paper microfluidics. In these techniques, all titration steps are installed on a single piece of patterned paper. Hydrophilic channels are created on the paper substrate in a flower (star)-like shape. Standard solutions and indicators are pre-deposited at the end of the channels. Titration is started by adding to the center of the device some microliters amount of sample solution, which follow through the channels, and reach to the standard solutions and the indicators [16,17].

Similar to paper-based electronic tongues, paper-based electronic noses have also found special interest in sensitive and selective detection of the volatile compounds emitted from different sources (foods, environment, or biological specimens) [18], [19], [20], [21]. For example, different paper-based optical nose devices have been fabricated to detect and identify volatile organic compounds emitted from human urine, blood, or exalted breath aiming to detect different kinds of cancer, non-invasively [22,23].

Besides to development of sensing and diagnostic devices based on a paper substrate, the paper has a long history in separation and chromatography. Cellulose-based supports became one of the popular stationary phases in separation techniques since the discovery of paper chromatography in the 1940s [24]. Paper is extremely cheap, biocompatible, easy to employ, store, and transport. In addition, its thinness and flexibility make it possible to be used in different designs. Moreover, chemical modification of the paper surface, due to the chemical reactivity of the hydroxyl groups on the paper surface, makes it an ideal candidate for preparing modified stationary phases. The wicking ability of paper/thread substrates eliminates the need for pumps to drive fluid flow. The possibility of the flow through capillary action in these substrates can simply reduce the device cost and also complexity.

Passive fluid transport in paper-based devices is controlled by the fabrication of hydrophobic barriers and hydrophilic channels. A wide range of paper patterning had been reported that can be classified into physical and chemical methods. Hydrophobic barriers can be patterned by physical methods such as wax patterning, plotting, inkjet etching, flexographic printing, stamping, cutting and shaping, screen printing, spraying, and drawing. As an alternative to physical methods, photolithography, plasma treatment, chemical vapor-phase deposition, and wet etching can be used for chemical paper hydrophobization [25], [26], [27]. Considering the advantages of paper chromatography and µPAD technology, their combination was introduced as one of the significant methods for separating various analytes. The separated species can be detected and determined by various analytical techniques such as electrochemistry, colorimetry, and mass spectroscopy.

µTADs have significant advantages such as high mechanical strength, flexibility, and high performance without needing any hydrophobic barrier and modification capability in wet conditions. Accordingly, attentions have been directed toward using thread as an alternative substrate to papers. Threads can be divided into two groups based on their preparation: Natural (cotton, wool, silk, linen, etc.) or artificial (nylon, polyester, polyurethane, polyethylene, polypropylene, acrylic, etc.). The production of natural threads is more complicated, time-consuming, and leads to irregular surface morphologies than manufactured threads with regular and smoother surfaces [28]. Both natural threads and artificial threads are applicable in designing µTADs. However, when selecting the type of thread, some parameters such as their surface chemistry, structure, and morphology should be considered. Similar to paper, the surface chemistry features of threads can be improved by modifications. Accordingly, µTADs have become one of the interesting substrates and stationary phases for separation due to their microfluidic properties, and modifiability, requiring small samples, reagents, or mobile phase. However, researches in microfluidic thread-based chromatography has not grown as paper-based devices did.

In this article, we will review the separation methods, most importantly chromatographic and electrophoresis, based on paper and thread. Our search was based on Scopus database and we focused on papers that published in 2015 or later. However, some of the previous articles, which were relevant to the topic of this review, were also considered. Since thread-based microfluidics share very similar experimental basis and analytical features with paper-based methods, we will review thread-based separation methods too. The review will begin with electrophoresis and then will be continued with chromatography. In recent years, special attention has been noted toward the separation of blood cells and blood plasma based on paper and thread microfluidics. Therefore, at the end of the article, recent advances in this important topic will be reviewed too.