Recent Advances and Applications in Paper-Based Devices for Point-of-Care Testing.

Point-of-care testing (POCT), as a portable and user-friendly technology, can obtain accurate test results immediately at the sampling point. Nowadays, microfluidic paper-based analysis devices (μPads) have attracted the eye of the public and accelerated the development of POCT. A variety of detection methods are combined with μPads to realize precise, rapid and sensitive POCT. This article mainly introduced the development of electrochemistry and optical detection methods on μPads for POCT and their applications on disease analysis, environmental monitoring and food control in the past 5 years. Finally, the challenges and future development prospects of μPads for POCT were discussed. © The Nonferrous Metals Society of China 2022.

Introduction

Point-of-care testing (POCT) is a low-cost, user-friendly, and portable technology that uses fast and convenient analytical instruments to obtain test results immediately at the sampling point [1]. By using low-volume of samples, POCT can be realized in hospitals, clinics, doctor's offices or homes [2]. Compared to central laboratory testing [3], POCT system has advantages of immediate turn-around time, easy-to-use format, high sensitivity and accuracy.

Nowadays, the technological challenge in the field of microfluidic paper-based analytical devices (μPads) is the main support for POCT systems. μPads are also known as lab-on-a-chip (LOC), which are proposed by Whitesides’ group in 2007 [4]. It miniaturizes the principal use of chemistry, biology and other laboratories to a small space of paper. It is an analytical platform that integrates the function of injection [5], reaction [6], separation [7] and detection [8] into paper by building hydrophilic and hydrophobic channels. The sample and reaction solution are driven by the capillary force of paper. μPads have the advantage of low production cost, simple method, easy processing, good biocompatibility, and small reagent consumption. Then the development of μPads has shown exponential growth in recent years [9].

μPads have been prepared by a variety of methods, such as photoetching[10, 11], inkjet printing [12], wax printing [13], laser processing [14], plasma processing [15], cutting [16], one-step plotting technology [17], flexographic printing [18], and stereoscopic printing [19]. We all know that hydroxyl groups on the paper are simple to be modified [20, 21]. So hydrophilic and hydrophobic regions can be easily constructed on the paper surface. Then the paper permeability and surface reaction activity are changed to create reaction channels for the migration, storage and reaction of reagents [22]. The μPads prepared by these advanced fabrication methods greatly expand the potential applications because paper can be used as the fine substrate for POCT devices. High-throughput determination of the content of multiple components in samples can be realized on μPads. Also, μPads provide a good platform for sample pretreatment, reagent transportation, mixing, separation and detection and other analytical functions [23-25].

As fast response rate and high sensitivity are the main demands for POCT, the detection system is vital for signal acquisition. So far, various detection technologies such as electrochemistry (EC), electrochemiluminescence (ECL), colorimetry, fluorescence (FL), surface-enhanced Raman scattering (SERS) and chemiluminescence (CL) have been assembled on μPads [26]. Honestly, each detection method has its advantages and drawbacks. Some optical detection methods use light sources as delivery or collection media to obtain signals. Because of acceptable sensitivity and response speed, these optical methods have become potential candidate technologies for μPads [27]. While the size of optical equipments such as lasers, spectrophotometers, charge-coupled devices (CCDs) and photomultiplier tubes (PMTs) make it difficult to integrate on μPads. Then the application of optical methods for POCT is relatively limited [28]. Compared with optical detection, electrochemical methods can get rid of the dependence on optical-based techniques. Through selection of electrode material and electrochemical technique, electrochemical detection was realized with fast response and high sensitivity [29]. Nowadays, to enrich these detection methods and achieve sensitive and accurate signal out-put, various micro- and nano-materials with different signal transduction mechanisms, such as metal nanoparticles [30, 31], metal oxide [32, 33], graphene or graphene oxide [34, 35], quantum dots [36, 37], hybrid materials [38, 39] and metal–organic frameworks [40-42] have brought new breakthroughs in the design of new paper-based sensors. In addition, signal readers tend to be miniaturized. Optical and electric signals can be read-out by portable devices like smartphone and electric watch [43, 44]. Therefore, combined with multiple detection technologies based on diverse sensing materials and portable signal readers, the POCT was realized on μPads and applied in the fields of disease analysis [45-48] (biological fluids like whole blood, serum, sweat, tears, urine, saliva, cells, viruses), environmental monitoring [49, 50] (water, gas, soil) and food control [51].

Therefore, considering that detection methods are crucial for paper-based POCT devices, it is essential to review and compare different existing detection methods on μPads. This article mainly introduces the development in the integration of detection methods on μPads in the past 5 years. Various detection methods including EC, ECL, colorimetry, FL, SERS and CL are applied on disease analysis, environmental monitoring and food control field. Moreover, the advantages and disadvantages of these optical detection methods are compared. Finally, the challenges and future development prospects of paper-based analytical devices for POCT are discussed. Although the portability of μPads makes them widely used, there is still a great room for improvement in stability and detection sensitivity.

Electrochemical Detection Methods

Electrochemical Method

Electrochemical (EC) method has been widely used to convert a biological or chemical event to an electronic signal. This detection method has been reported to integrate on the μPads by Henry’s group [29]. EC combined with μPads is known as electrochemical paper-based analytical devices (ePads). EPads are always sensitive and have quick response, which have been a main support at the POCT. Recent examples of applications are reviewed here to demonstrate the potential of ePads in environmental monitoring [49] and biomedical analysis [52-78] as well as food safety control [79-81] field.

Environmental Monitoring

Metal ions have been measured by ePads in Silva-Neto’s report [49]. A "plug-and-play" (PnP) assembly for multiplexed detection of Fe, Ni, Cu, Zn, Cd and Pb in river water samples with screen-printed ePad was described. The device had good selectivity and aspirated sample volume can be managed well. The detection values for these metal ions were in the range of 0.9–10.5 μg/L.

Biochemical Assays

As reported by Liang [52], a wearable electrochemical sensor using three-dimensional paper-based microfluidic electrochemical device (3D-PMED) for real-time monitoring of potassium ion (K+) in sweat was fabricated. The 3D-PMED integrated a screen-printed K+-selective sensor with limit of detection (LOD) of 132 mmol/L. Also, per decade of K+ for the electrode response potential was 61.79 mV. In 2017, a parylene C-coated newspaper (PC-paper) was developed by patterning of metal layers. These chemically stable electrochemical platforms were applied to the detection of electrolyte cations, like H+ and K+ [53]. EC method was used for investigating the fluid dynamics. Such as a 2-layer μPad was used for increasing the flow rate through precise control of the channel height. A ferrocene complex was analyzed and anodic stripping detection of cadmium with fivefold enhancement signal was performed on this ePad [54].

Based on electrochemical methods, small molecules can also be detected on μPads. For example, as reported by Ming’s work [55], 17β-estradiol (E2) was detected by a folding aptasensor platform with the label-free electrochemical detection method. Amine-functionalized single-walled carbon nanotube/ new methylene blue/ AuNPs were adopted for immobilizing the aptamer. The calibration curve showed a linear range from 10 pg/mL to 500 ng/mL and a LOD of 5 pg/mL. In 2018, Sales and his team have fabricated an ePad by applying the homemade conductive inks for structuring the electrodes. Square wave voltammetry (SWV) method was used to detect 3-nitrotyrosine (3-NT). As for the sensitivity of the sensor, a low LOD of 49.2 nmol/L of 3-NT can be obtained [56]. In Wang’s work, they reported a paper-supported photoelectrochemical sensing platform based on surface plasmon resonance enhancement for real-time H2S determination. H2S can induce surface plasmon resonance (SPR) enhancement between Ag NPs and CdS QDs [57].

There are also some works about glucose detection [58-62]. Chaiyo’s group have introduced an ePad for the non-enzymatic detection of glucose in honey, white wine and human serum. The screen-printed carbon electrode was modified by cobalt phthalocyanine, grapheme and an ionic liquid (CoPc/G/IL/SPCE). The modified electrode on ePads had excellent electrocatalytic activity towards glucose in a wide calibration curve [58]. Glucose can also be detected by a wearable platinum sensor in Sarwar team’s work [59]. As reported by Cinti’s group [60], the filter paper was used as a container for reactions. Prussian Blue Nanoparticles (PBNPs) were produced on filter paper and then a reagentless electrochemical point-of-care device using glucose oxidase for glucose detection was developed with the concentration ranging up to 25 mmol/L (450 mg/dL). Cellulose nanofibers (CNs) were performed on ePad for glucose measurement in blood samples [61], as shown in Fig. 1a. First, the electrospinning method was used for preparing cellulose acetate (CA) nanofibers. Then, in alkaline solution, the paper layer was changed to cellulose by deacetylation. The paper was treated with trimethyl chitosan (TMC) to obtain a smooth and continuous CNs layer. A thick layer of Au was sputtered on the TMC/CNs substrate and then reduced graphene oxide (rGO) was used to modify the working electrode. At last, the immobilization of glucose oxidase was performed on the CNs layer. The ePad has a linear range of 3.3–27.7 mmol/L for glucose with a LOD of 0.1 mmol/L. By converting electrochemical signals into optical readouts, Xu’s group showed a closed bipolar electrode (CBE)-based two-cell electrochromic device for detection of lactate, glucose and uric acid [62]. A specific oxidase was coupled to the analytical cell color change, which is related to the concentration of metabolites.

Fig. 1

Some examples for EC detection. a Schematics of glucose-ePAD with different fabrication method for glucose detection [61]; b Schematic illustration of screen printed carbon electrodes [70]; c Fabrication and modification process of the multi-parameter ePAD for the detection of CEA and NSE [73]; d Illustration of the whole procedures and sensing principle for OTA determination [79]

In Li’s review [63], they introduced the types of neurotransmitters and biological sample sources which were used for neurotransmitter detection and then reviewed the traditional fabrication technologies and modification methods for paper-based electrochemical POCT devices. In Lu’s work [64], ePad was used for human immunodeficiency virus (HIV) DNA detection with methylene blue (MB) as a redox indicator. A paper-based electrode was made by using nickel metal–organic framework (Ni-MOF) composite/Au nanoparticles/carbon nanotubes/polyvinyl alcohol (Ni–Au composite/CNT/PVA). Ni–Au composite/CNT/PVA can achieve interactions between MOF and single-stranded DNA. Then a higher loading of the probe DNA was made. Peak current varied with the concentration of HIV DNA. The device sensed well in a linear range of 10 nmol/L–1 μmol/L and a low detection limit of 0.13 nmol/L. Narang et al. fabricated an ePad combined with Zn–Ag nanoblooms to detect herpes [65]. In infected patient samples, the ePad showed optimum current response in two linear ranges of 113–103 and 3 × 105–106 copies/mL with LOD of 97 copies/mL. In Cinti’s work [66], printed electrochemical platforms were performed for ssDNA and dsDNA detection. The methylene blue (MB)-tagged TFO probes were coated on the working electrode. Then, TFO probes were fabricated on ePad and then dsDNA sequence can be detected in serum samples with the LOD of 7 nmol/L.

In recent years, immunoassay has been used for the detection of antigens such as biomarkers. For example, in 2019, Qi’s [67] team synthesized in-situ molecularly imprinted polymers (MIPs) on movable valve microfluidic paper-based electrochemical device (Bio-MIP-ePADs) for clinical detection of carcinoembryonic antigen (CEA) based on the strategy of antibody-free biomarker analysis with the detection range of 1.0–500.0 ng/mL, and the detection limit could be achieved at 0.32 ng/mL. Kaushik’s group [68] proposed an electrochemical immunosensing platform for Ebola virus (EBOV) detection at pmol/L concentration within 40 min. It was a cost-effective, rapid, sensitive and selective sensor to detect Ebola virus disease (EVD) at point-of-care (POC). Cao’s group [69] developed a sensitive immune method for human chorionic gonadotropin (HCG) detection on paper-based microfluidic device. Alkaline phosphatase combined secondary antibody (ALP-IgG) with functionalized gold nanoparticles was used as the signal antibody label. The hydrophilic test zones of the aldehyde-functionalized screen-printed electrodes (SPEs) were biofunctionalized with capture antibodies (Ab1). And the LOD of human chorionic gonadotropin (HCG) was 0.36 mIU/mL. Honikel’s group [70] provided a paper-based sensing platform by immobilizing different antibodies (antilactoferrin (Lfn) or anti-immunoglobulin E (IgE)) onto screen-printed carbon electrodes. The LODs were 0.05 mg/mL and 40 ng/mL for Lfn and IgE, respectively (Fig. 1b). Li’s team [71] developed a microfluidic paper-based biosensor integrated with zinc oxide nanowires (ZnO NWs) for rabbit immunoglobulin G (IgG) and the immunodeficiency virus p24 antigen detection. The whole procedure just took less than 25 min. The ePad was performed for detecting rabbit immunoglobulin G (IgG) in phosphate-buffered saline with the LOD of 60 fg/mL and the immunodeficiency virus p24 antigen in human serum with the LOD of 300 fg/mL. Wang’s group [72] developed a label-free paper-based electrochemical immunosensor by using screen-printed working electrode (SPWE) to detect carcinoembryonic antigen (CEA). Amino functional grapheme (NH2-G)/thionine (Thi)/gold nanoparticles (AuNPs) nanocomposites were synthesized to raise the detection sensitivity. In 2019, Wang’s team [73] fabricated the paper-based device by wax printing and screen-printing method. The device enabled the functions of sample filtration and auto injection. Amino functional graphene (NG)-Thionin (THI)- gold nanoparticles (AuNPs) and Prussian blue (PB)- poly (3,4- ethylenedioxythiophene) (PEDOT)- AuNPs nanocomposites were synthesized to modify the working electrodes not only for promoting the electron transfer rate, but also for immobilization of the CEA and NSE aptamers. A multi-parameter aptasensor on ePad for simultaneous detection of CEA and neuron-specific enolase (NSE) in a clinical sample was established. The ePad exhibited good linearity in ranges of 0.01–500 ng/mL for CEA and 0.05–500 ng/mL for NSE, respectively (Fig. 1c). Micropipette-tip immunoelectroanalytical platform coupled with staple-based paper device was established. Anti-tissue transglutaminase was detected with immunoassays performing in the polypropylene micropipette tips [74]. The platform was very promising for decentralized analysis. Besides, Zheng’s group [75] fabricated a porous structure of AuNP-modified paper working electrode (Au-PWE) as a sensor substrate with a feature of all-round conductivity and plenty of active sites favoring biological ligand attachment, which was used to detect CEA and prostate-specific antigen (PSA) in enzyme-free condition. Wei’s group [76] fabricated gold nanoparticles (AuNPs)/reduced graphene oxide (rGO)/thionine (THI) nano composites as working electrodes for sensitive detection of PSA. THI was used as the electrochemical mediator to transduce the biological recognition between DNA aptamer and PSA. The linear range for PSA was 0.05–200 ng/mL with the LOD of 10 pg/mL.

Additionally, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a global pandemic outbreak in 2019. Yakoh’s group developed a label-free paper-based electrochemical immunosensor for immunoglobulin detection against SARS-CoV-2 without the specific requirement of an antibody [77]. The principle was that the presence of SARS-CoV-2 antibodies would interrupt the redox conversion of the redox indicator. Then the signal decreased. This sensor was proven to be effective in real clinical sera from patients. West Nile virus can be measured with a LOD of 10.2 particles in 50 μL of cell culture media by Channon’s group [78] on an ePad with Au electrodes.

Food Safety Control

In Zhang’s work [79], Ochratoxin A (OTA) was used as the model for an ePad immunoassay. Functionalized MoS2-Au@Pt (Ch-MoS2-Au@Pt) was produced to immobilize label aptamer (apta2) for signal amplification. The Ch-MoS2-Au@Pt-apta2 had the function of specific biorecognition and can be the catalyst for H2O2 reduction reaction. Then EC signal can be produced on ePad. As hydroxyl radicals can be produced in the reaction and induce TMB to change color, a colorimetric method was also established for OTA detection. So the dual-mode detection for OTA was obtained in the linear ranges of 0.1–200 ng/mL and 200- 1 × 10–4 ng/mL for visual and EC detection, respectively (Fig. 1d). DNA purification testing was performed on a micro ePad for foodborne pathogens detection. The whole procedure can be performed in half an hour and Escherichia was successfully detected [80]. Writing fabrication method on ePad was reported by Li’s group [81]. The writing can be performed for the microfluidic channels and electrodes by two commercial pens. The hydrophobic part was written by a wax pen and electrodes were produced by a conductive-ink pen. The writing ePad was used to detect Salmonella typhimurium DNA by dual mode methods (colometry and EC), LODs of 1 nmol/L and 1 mmol/L, respectively.

Eelectrochemiluminescence (ECL)

In order to maintain the advantages of paper devices, such as easy qualification and development, suitable detection techniques are required [82]. ECL is one of the most versatile analytical methods, due to its high sensitivity and signal-to-noise ratio. As examples, recent works have been performed on paper-based ECL devices to detect miRNA. Zhou et al. presented a portable ECL chip driven by CRISPR/Cas13a, which could be activated by target miRNA. Then, it triggered the subsequent exponential amplification with LOD as low as 1 × 10–15 mol/L of miRNA-17 [83]. Tumor cells can cause different degrees of harm to the human body. Therefore, the rapid detection of tumor cells using paper-based devices is important for clinical diagnosis. In Ge’s work, AuPd nanoparticles (NPs) were used as a carrier and catalyst for luminol-H2O2 system. With the releasing of H2O2 from target cells, MCF-7 can be detected in the range of 1.5 × 102–2.0 × 107 cells/mL and LOD of 40 cells/mL [84] (Fig. 2a). Similarly, Yang’s work used semicarbazide and nano-silver as dual enhancers, and multi-branched double-stranded DNA nanowires (MBdsDNA) as carriers to detect tumor cells MCF-7, CCRF-CEM, HeLa, and K562 [85]. MCF-7’s detection range and LOD were similar to that of Ge’s work. Besides, paper-based ECL devices can be used for analysis of metal ions. As reported by Huang’s work, due to Ni2+ and Hg2+ have quenching effects on ABEI’s ECL emission, they first made an ECL sensor with repeated automatic cleaning of the working electrode to detect heavy metals [86] (Fig. 2b). The detection of cancer biomarkers can make judgments on course of disease, the existence and prognosis of the tumor cells. Sun has developed a rotating μPad for multi-step ECL immunoassay of CEA and Prostate specific antigen (PSA) with the advantages of reusable rotary valve and short response time [87]. In addition, based on the DNA Walker's strand displacement reaction and the catalysis of DNA-Pt/CuTNFs [88], an enhanced luminol ECL signal was obtained to detect streptavidin with a low detection limit of 33.4 fmol/L (Fig. 2c). A diode was coupled on an ePad, which can dramatically enhance the signal intensity in Qi’s work [89]. By using gold electrode array and an electromagnetic receiver coil, the ECL for detection of H2O2 can be on a par with photomultiplier tube (PMT)-based results. The high sensitivity with the linear range of 10 nmol/L to 1 mmol/L was obtained. Moreover, paper-based ECL devices could be used for analysis of organic and inorganic compounds and other substances [90-103] involving various scientific fields such as environment monitoring, biochemical assay, food safety, and so on (as shown in Table 1). Then the paper-based microfluidic system [104] has great application potentials.

Fig. 2

Some examples for ECL detection. a Paper-based ECL device for MCF-7 detection [84]; b Schematic illustration for paper-based ECL device for Ni2+ and Hg2+ detection [86]; c Principle for paper-based ECL device of the analyte-triggered DNA walker [88]

Table 1

Summary of the corresponding ECL applications on μPads in the fields of the environment, food, biochemistry in past 5 years

Materials	Target molecule	Samples	Advantages	LOD	Refs.
CdTe QDs-H2, Au@g-C3N4, NSs-DNA1 and carboxylated Fe3O4 magnetic nanoparticles	MiRNA-155 and miRNA-126	–	Favorable linear response and excellent sensitivity	5.7 fmol/L and 4.2 fmol/L	[90]
DNA (S1)-AuPd NPs	miRNA-155	–	Acceptable specificity and favorable stability	0.67 pmol/L	[91]
GQDs load surface villous Au nanocages	CA153	MCF-7 cell	Low-cost and fast	0.0014 U/mL	[92]
Au@Pd nanoparticles and Pt-Ni alloy particles	MCF-7 cell	MCF-7 cancer cells	In-situ screening of anticancer drugs and monitoring the number of apoptotic cancer cells	300 cells/mL	[93]
Three separated arrays of reservoirs	HL-60 cancer cells	HL-60 cancer cells	Distinguish the tumor cells from normal cells	80 cells/mL	[94]
A bipolar electrode array	MCF-7 cell	MCF-7 cell	Simple and suitable for high-throughput detection	52 cells/mL	[95]
HRP functionalized Au nanocubes	Pb2 +	Lake water	Portable, low-cost and high efficiency	0.52 nmol/L	[96]
PFCeO2 NPs and 50 nm Ag NPs	Pb2 +	Mineral water	A wide linear range, good selectivity and reproducibility	0.016 nmol/L	[97]
Green-luminescent N-GQDs	α-fetoprotein	Human serum	A wide calibration range, good specificity	1.2 pg/mL	[98]
Magnet-controlled self-circulating chip	Circulating tumour nucleic acids (CTNAs) in serum clinical CTNA samples	Blood samples	Highly efficient signal generation and desirable specificity	100 amol/L	[99]
Graphite paper, Pt NPs, chitosan-multi-walled carbon nanotubes (CS-MWCNTs) and Au@Pt nanostructures	H2O2 CEA	Human serum sample	High selectivity, a wide linear range, good reproducibility	0.5 µmol/L (S/N = 3) of H2O2 5.0 pg/mL for CEA	[100]
Silica nanochannel-assisted electrode	Alkaloidal drugs	Buffers and human serum	Flexibility and universality	1.799 nmol/L and 11.43 mol/L	[101]
Bipolar electrodes	Glucose, lactate and cholinc	Human serum	Simple, efficient and versatile	7.57 μmol/L, 8.25 μmol/L and 43.19 μmol/L	[102]
Bipolar electrodes	pathogenic DNAs	–	High sensitivity and multiplexed analysis	0.1 fmol/L	[103]

However, the above electrochemical method needs electrode couples on µPad. Electrode fabrication is a crucial step to fabricate. The power should be added on the device. Some electrodes which were used on µPad still cannot have the performance in comparison to conventional metallic electrodes. Some other optical detection methods are shown below for µPad.

Optical Detection Methods

Colorimetric Detection

μPads with simple user interpretation and instruments are desired for POCT. Colorimetric detection is the primary technique applied in μPads, because color intensity can be realized easily by an ultraviolet–visible (UV–vis) spectrophotometer. Until now, it is widely applied in analysis of inorganic ions [105-114], biomedicals and proteins [115-122], nucleic acid and drugs [123-136], etc.

Colorimetric detection gets great potential applications in environmental monitoring. Generally, direct colorimetric detection can be measured by comparing the color intensity of the reaction spots with the standard colors [137]. For example, Cu2+ reacted with 3-(5-hydroxy-4-carboxyphenylimino)-5-fluoroindol-2(H)one (HCFI) reagent to obtain a colored complex. Then a miniaturized spots patterned commercial book-paper was developed for Cu2+ detection as low as 1 × 10−3 μg/mL [105]. In 2019, a silver triangular nanoplate (AgTNP)-modified paper strip was selectively used for detection of iodine. The interaction between AgTNPs and iodine [138], changed the color from blue to white and the LOD for iodine was 7 μg/L.

Besides, colorimetric measurement based on distance is a distinctively visual quantitative method. The colored length on μPad has the relation to the concentration of targets [139, 140]. For instance, a distance-based method [107] for Hg2+ testing was developed. A precipitated tetramethylbenzidine (TMB) was immobilized on paper chip. When DNAzymes reacted with Hg2+, the G-quadruplex-hemin DNAzymes was formed and a color band was generated (Fig. 3a). A trace concentration of 0.23 nmol/L for Hg2+ was detected.

Fig. 3

Some examples for colorimetric detection. a Distance assay for Hg2+ by using G-quadruplex DNAzyme [107]; b Illustration of enzyme-inorganic hybrid nanomaterials synthesized on paper chips [115]; c Schematic diagram for ring-oven washing procedure [121]; d Illustration of distance detection for CEA biomarker [141]; e Design of the CRISPR/Cas9-mediated TL- lateral flow strip. [127]

Biochemical Analysis

As for biomedical analysis, a visual colorimetric μPads [115] was developed by the in situ synthesis of a hybrid functional material, GOx@Mn3(PO4)2. The content of glucose in biological samples can be detected with LOD of 0.01 mmol/L (Fig. 3b). Proteins, like enzymes and antigens, were also detected by μPADs. For example, Gong’s team [116] developed a microfluidic platform that collected human serum by a pulling-force spinning top (PFST) and paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) for quantity of IgA/IgM/IgG in an instrument-free way. It can easily isolate the serum without any clinical apparatus, and a portable smart phone made it easy to record the intensity signal. Moreover, in 2020, Li’s group[117] presented a microfluidic system that can centrifugate human whole blood and quantify carcinoembryonic antigen and alpha fetoprotein by ELISA method with LODs of 360 pg/mL and 280 pg/mL for CEA and AFP, respectively. Chandra [120] detected alkaline phosphatase (ALP) using colorimetric method on µPad by antibody immobilization on paper surface. The LOD for the ALP detection was 0.87 (± 0.07) U/mL. To wash the nonspecific-binding antibody from the paper surface, a novel continuous washing strategy with ring-oven was established by our team [121]. To verify the washing results, HRP-catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 colorimetric system was used for CEA detection with the lower LOD of 0.03 ng/mL (Fig. 3c). Moreover, the detection of nucleic acids can also be carried out on paper with super low LOD. Shu’s group [123] has developed a micro-patterned paper device (μPPD)-based colorimetric strategy for double-stranded DNA (dsDNA) detection by using polydiacetylene (PDA) vesicles. The quantitative analysis of the target can be down to 10 nmol/L. By using dye-based reaction, Goswami [124] reported a colorimetric method for pan malaria and P. falciparum species detection with LODs of 61.50 ± 6.43 pmol/L for PLDH and 63.97 ± 7.24 pmol/L for Pf GDH, respectively. In 2019, Chen’s team [125] has developed a rapid and sensitive colorimetric sensing platform for the detection of ketamine. By using competitive ELISA test on a μPad, the results can be obtained within 6 min with the LOD of 0.03 ng/mL.

Furthermore, a distance-based paper analytical device (dPAD) [126] was fabricated to realize the loop-mediated isothermal amplification (LAMP) and semiquantitative determination of genomic DNA in E. coli as low as 4.14 × 103 copies/μL. For immunoassay, CEA was semiqualitative detected by our team with distance-based colorimetry [141]. With the precipitated TMB-H2O2 added on the paper-based device, LOD of 2 ng/mL can be obtained with a visible bar (Fig. 3d). In 2021, a non-immunoassay dPAD was introduced for the detection of cardiac troponin I (TnI) [142]. Without any external blood separation, TnI in whole blood samples was determined by using the dPAD with the LOD of 0.025 ng/mL.

In the field of biochemical analysis, the lateral flow assay (LFA) [143-147] is another common visual platform. Targets usually are induced by lateral capillary force and then react with the biorecognition molecules which are bonded on the porous membrane surface. The results can be read out via colored molecule-labeled biorecognition molecules. LFA is a potential candidate for POCT because of simple operation and one-step analysis procedure. For example, Hou’s team [146] has developed a LFA for the simultaneous detection of glucose and glycation ratios in human serum albumin. In 2021, an ultrasensitive LFA [147] was introduced for the determination of the telomerase activity. With the deblocking of ssDNase activity of CRISPR/Cas12a by telomerase extends activators, the telomerase activity was detected as low as 57 cells/mL within 1 h. In 2020, as SARS-CoV-2 was spread around the world, LFA [127, 148] was regarded as the most efficient way to realize POCT. A triple-line lateral flow assay (TL-LFA) for the dual-gene detection of SARS-CoV-2 was established [127]. With the CRISPR/Cas9 mediating, multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) was realized. The genes of envelope (E) and open reading frame lab (Orf1ab) were detected from the RNA standards in cell-cultured SARS-CoV-2 and SARS-CoV-2 viral. The LOD was 100 RNA copies of 25 μL reaction (Fig. 3e). Furthermore, other DNA analysis using LFA were reported [128, 129]. Cui’s team has developed a tetra-primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR)-LFA for the detection of two alleles [125] within 75 min. Jauset-Rubio [129] has reported a LFA for the multi-channel detection of DNA in Francisella tularensis and Yersinia pestis. Using isothermal recombinase polymerase amplification, the assay results were obtained within 1 h and the LOD was 243 fg for Francisella tularensis and 4 fg for Yersinia pestis, respectively.

For food safety, colormetry method can measure the content of toxin [149-151] and drugs [152, 153]. A single-line-based LFA (sLFA) strip [150] without the control line was explored for aflatoxin B1 determination. In this assay, an orthogonal emissive upconversion nanoparticle (UCNP) served as a signal substance and calibrator, which had emission at two wavelengths. Zhou’s group [152] has developed a LFA with up-converting phosphor method for the determination of morphine and methamphetamine with LOD of 5 ng/mL for morphine and 10 ng/mL for methamphetamine.

Fluorescence (FL) Detection

Fluorescence (FL) is the emission after fluorophores or fluorescent dyes excitated by an energy at certain wavelength. For μPads, FL has been a main optical method with high sensitivity and high selectivity [154].

In the field of environmental monitoring, FL was applied on detection of metal ions [155-162], anion [163-165], gas [155], and organic compounds [166-168]. Liu [155] has reported a FL probe on μPads with carbazole for the detection of Cu2+ and gaseous H2S. In 2019, a paper-based platform was established [157] using a hand-held UV lamp as an excitation resource. For Cd2+ detection, the FL emission signal can be captured by a mobile phone. Exploiting thin-shell CuInS2@ZnS QDs, Cd2+ was measured even at a trace concentration of 105.86 nmol/L. Moreover, a μPads was demonstrated for F− detection with the fluorescence resonance energy transfer (FRET) method [163]. The linear range for F− was 0.05–0.55 nmol/L, with a LOD of 9.07 pmol/L (Fig. 4a). In 2020 [166], taking advantage of the blue luminescence of graphene quantum dots (GQDs), o- and p-nitrophenols (ONP and PNP, respectively), two kinds of endocrine disruptors were determined selectively and sensitively. The quantitative analysis of ONP and PNP showed linear ranges of 0.30–60.0 µg/mL and 0.20–40.0 µg/mL, respectively, with LODs of 0.07 µg/mL for ONP and 0.03 µg/mL for PNP.

Fig. 4

Some examples for FL detection. a Fluorescence “off-to-on” mechanism on the GO paper for F− detecting [163]; b Schematic illustration of the tricolor probe for Cu2+ [169]; c Illustration of distance-dependent immunoassay on μPAD [177]; d Schematic illustration for FL detection of CEA on μPAD [182]; e Schematic illustration of portable test strips and wearable devices for the analysis of TC [199]; f Schematic illustrations of a single dual-emissive ratiometric probe for thiram by smartphone [203]

As FL molecules can be used as signal probes, FL has many applications in inbiochemical studies. For instance, μPads with different carbon and quantum dots [169] were reported. Tricolor FL probe was established with the addition of different concentrations of Cu2+. For Cu2+, the LOD was 1.3 nmol/L in human urine (Fig. 4b). Furthermore, a highly ratiometric fluorescent N, S co-doped carbon dots (N,S-CDs) probe towards ClO− [170] detection had been applied to paper-based devices. The N,S-CDs probe showed excellent linearity in the range of 0.067–60 μmol/L with a LOD of 9.1 nmol/L.

In addition, μPads have been used for the rapid detection of chemical molecules (e.g. glucose, dipicolinate (PDA) and so on [171-175]. On the basic of an Eu (III)-EDTA complexes functionalized poly(diacetylene acid) derived liposomes, a novel ratiometric FL detection system was established on a paper chip for the visual detection of PDA [171]. In 2020, Golmohammadi [175] has reported a cellulose-based wearable patch for sweat biomarker detection. Glucose, lactate, pH, chloride, and volume can all be read out by a smartphone-based FL imaging module. A smartphone APP was also designed in that work.

Besides, μPads have measured various proteins (enzymes and antibodies) in whole blood, urine, saliva and sweat, as they are regarded as biomarkers of some diseases [176-181]. Lin’s group has described a paper-based immunoassay for the matrix metalloproteinase-7 (MMP7) detection, which was corresponding to renal cancer [177]. Based on sandwich-type immunoreaction, silver nanoparticles (AgNPs) were first labeled with secondary antibodies. After immunoassay, the signal antibody with silver nanoparticles was dissolved in nitric acid to produce Ag+. CdTe quantum dot was firstly physically adsorped on the nitrocellulose membrane. Due to quenching effect Ag+, the distances can produce on the paper-based chip under 365 nm shining. With the concentration of MMP7 increased, the quenching distance increased and the LOD was as low as 7.3 pg/mL (Fig. 4c). Besides, enzyme activity also can be detected by μPads [179-181]. A λ exonuclease-assisted paper-based FL assay [179] was described for facile testing of polynucleotide kinase (PNK) activity by fluorescence intensity on paper surfaces, achieving sensitivity of PNK activity down to 0.0001 U/mL. In addition, paper-based FL immunoassays were used commonly for the detection of antibody biomarker [182-185]. CdTe/CdSe QD and relevant enzyme were saturated on paper. Also, DNA-gated mesoporous silica nanocontainers (MSNs) were combined. Then the FL detection of CEA was realized with a low LOD of 6.7 pg/mL [182] (Fig. 4d).

Furthermore, since nucleic acids are one of the most fundamental biological substances in all organisms [186], the accurate measurement becomes a commom concern for disease diagnosis. Lu [187] has introduced a paper-based sensor system for a nucleic acid amplification test with an internet platform. The paper-based sensor enabled genomic DNA’s identification for Escherichia coli and Campylobacter jejuni, with the LOD of 2 × 103 copies/μL. Other μPads for nucleic acid detection [188-192] have been summarized in Table 2. Another important application for μPads is to directly detect cells. In 2020, a fluorescence method was established on a dual-layer paper microfluidic chip for the detection of ROR1 + [193]. A smartphone-based FL microscope and automated image processing were established to enumerate particles, with the LOD of 1 cell/μL.

Table 2

Summary of the corresponding applications on μPads with FL detection method in the past 5 years

Materials	Fabrication method	Detection modes	Target molecule	LOD	Samples	Refs.
Red quantum dots and cyan carbon dots (CDs)	Jet-printing with filter paper	The quenching of red FL through the formation of dispersive QDs aggregates	As(III)	5 ppb	Tap water and lake water	[156]
Graphene oxide (GO) sheet	Adding the aptamer solution and GO solution onto the square shape paper cutted by a craft punch	The FL quenching property of GO sheet by Pb2+ through the FŐrster Resonance Energy Transfer (FRET) process	Pb2+	0.5 pmol/L	Tap water, lake water, milk, and human blood serum	[158]
Thiomalic acid bonded to CdTe (TMA-capped CdTe)	Paper immobilized with TMA-capped CdTe	The FL quenching of the reaction of red-emission TMA-capped CdTe with Ag+ by electrostatic interaction	Ag+	13.16 nmol/L	Human plasma, bovine serum, lake water, and green tea water	[159]
Carbon nanodots (CDs)	Printing method	The FL turn-off assay with varying binding properties of CDs by various metal ions	Pb2+ and Cu2+	Pb2+, 0.12 μmol/L; Cu2+, 0.076 μmol/L	Pearl River	[160]
Rhodamine B	Eyeliner pencil method	The FL quenching of rhodamine B by formation of RB-Au3+ complex	Au3+	0.15 mg/L	Ore samples	[161]
1-Thio-β-ɒ-glucose bonded to copper nanocluster (TG-CuNCs)	Cutting method	The FL quenching of the reaction between Hg2+/ S2− and TG-CuNCs	Hg2+ or S2−	1.7 nmol/L and 1.02 nmol/L	Pond and river water	[162]
Europium tetrakis dibenzoylmethide triethylammonium (EuD4TEA) and gold nanoparticles (Au NPs)	Impregnating filter paper into the mixture of EuD4TEA and Au NPs	The FL turn-on cyanide (CN−) assay	CN−	1 × 10–2 mol/L	Drinking water	[164]
Aminomodified graphene oxide (GO-NH2) with silicon coated rhodamine B (RBDS) nanospheres	Dripping the mixture solution of poly (vinyl alcohol) (PVA-1788) and RBDS/GO-NH2 nanosensor solution onto a common filter paper	The distinguishable fluorescent color change by GO-NH2 with the oxidation of hypochlorous acid	HOCl	2.92 μmol/L	DI water, tap water, East Lake water and Yangtze River water	[165]
Amino groups linked-carbon quantum dots (CDs@NH2)	Dripping the mixture solution of poly (vinyl alcohol) (PVA-1788) and CDs@NH2 solution onto a filter paper	The FL quenching of CDs	TNT	0.213 μmol/L	Ground water	[167]
Papain-stabilized gold nanoclusters (papain-AuNCs)	Papain-AuNCs solution scattered the test strip	Papain-AuNCs as the FL probe	Glyphosate (Glyp)	0.035 ng/ mL	Tap water	[168]
Functionalized manganese-doped carbon dots (FMn-CDs)	Dropping FMn-CDs onto a circular fiber filter paper	A ratiometric FL biosensor with Eu(III)	2, 6-dipicolinic acid (DPA)	1 μmol/L	Lake water, River water and Fetal bovine serum (FBS)	[172]
Nano zinc 5, 10, 15, 20-tetra(4-pyridyl)-21H-23H-porphine (nano ZnTPyP)-CdTe QDs	Cutting method	The FL response between nano ZnTPyP-CdTe QDs and caffeine	Caffeine	1.53 × 10–11 mol/L	Water, human plasma, cell culture fluid	[173]
CDs	Soaking a filter paper in CDs	FL of reaction between the affluent amino groups on CDs and nitrophenols	Nitrophenols (3-nitrophenol and 4-nitrophenol)	0.5 mmol/L and 0.1 mmol/L	HEPG-2 cells	[174]
Rox-DNA functionalized quantum dots	Immersing filter paper to make the paper functional	The FL color changed from red to yellow-green	Telomerase activity	10 cells	Urine	[180]
CdTe QD bonded polythiophene (CP)	Wax-printing the design on paper	The aggregation induced emission enhancement (AIEE)of the interaction between CP and thiocholine	Cholinesterase activity	0.14 U/L	Human serum	[181]
CdTe QDs	inkjet printing method and ring-oven washing	FL signal enhancement by “Sandwich” immunoassay with CdTe QDs	Immunoglobulin G (IgG)	0.4 ng/mL	Human serum	[183]
NaYF4: Yb, Er upconversion nanoparticles (UCNPs)	One-step plotting method	FL Resonance energy transfer reaction	Immunoglobulin E (IgE)	0.13 IU/mL	Human serum	[184]
Hairpin strand 1 and hairpin strand 2 modified with the fluorophore FAM	Primary antibodies immobilized on the paper by chitosan	FL of AFP by triggered hybridization chain reaction labeled on detection antibody	Alpha-fetoprotein (AFP)	1.0 pg/mL	Human serum	[185]
A fluorogenic DNAzyme probe	Printing wax on various paper substrates	A fluorogenic DNAzyme probe	E. coli DNA	100 cells /mL	E. coli	[188]
Tetraphenylethene and benzothiadiazolemoieties (TPE-BTD)	Dropping TPE-BTD solution and PB containing dopamine, HRP and GOx onto the cellulose paper’s surface	The FL quenching effect of TPE-BTD	G-quadruplex DNA and Dam MTase	0.21 nmol/L and 0.016 μmol/L	Serum	[189]
3-aminopropyl trimethoxysilane (APTMS)	A simple one-step surface modification method using APTMS	The FL of Cy3-labeled Giardia amplicon	Giardia lamblia DNA	22 nmol/L	Giardia lamblia	[190]
Taqman probes	Printing by the wax printer	FL of duplex-specific nuclease (DSN) amplification	MicroRNAs (miRNAs)	miRNA-21 of 0.20 fmol/L and miRNA-31 of 0.50 fmol/L	Cancer cells of A549 and HeLa, and hepatocyte LO2	[191]
Labeled DNA probes-QDs	Spotting QD-DNA on the biotin modified papers	A ratiometric detection based on FRET from QD donors to dye molecules	Oligonucleotide	0.1 pmol	Full goat serum	[192]
Hydrophilic fluorescent hydrogel	Hg2+ immobilized with paper by polydopamine-based coating approach	A specific chemical reaction between Hg2+ and the thiourea moieties	Hg2+	1 × 10–7 mol/L	Water and food samples	[195]
Silicon nanoparticles (SiNPs) bonded to Eu(III) (SiNPs/Eu)	Cutting method	The changed FL emission by reaction of cyan with tetracyclines	Tetracyclines (TCs: chlortetracycline, tetracycline,doxycycline)	0.4 μmol/L	Honey and farmed fish	[200]
g-C3N4 nanosheets coupled with Eu3+	Immersing paper into g-C3N4/Eu3+	The enhancing effect of red FL of Eu3+ by TC through the antenna effect	Tetracycline (TC)	6.5 nmol/L	Milk	[201]
NaYF4:Yb/Tm upconversion nanoparticles with Cu2+	Immersing paper into NaYF4:Yb/Tm@ poly(acrylicacid)-Cu nanoprobe	The FL quenching effect with thiram on upconversion nanoparticles	Thiram	0.1 μmol/L	Apple juice	[202]
CdTe QDs and nano zinc 5, 10, 15, 20-tetra(4-pyridyl)-21H-23H-porphine (nano ZnTPyP)	Adding NAC-capped CdTe solutions on the circular paper	A “turn-off–on” FL mode of CdTe with carbamate pesticides	Three carbamate pesticides (metolcarb, carbofuran, and carbaryl)	0.91 μg/L	Apple, cabbage and tea water	[204]

Simple, rapid, and instrument-free quantitative detection is very vital for the effective food safety control [194]. Especially, inorganic content [195-197] in water and food is one of the concerns by modern citizens. For example, a μPad was designed for membraneless gas-separation and iodate determination by using the bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) [196]. Based on the fact that gold core of BSA-AuNCs was etched and the red emission was quenched, the iodate was monitored by fluorometric detection with LOD of 0.005 mmol/L. In 2019, a ratiometric fluorescent nanoprobe, label-free carbon dots (CDs), with dual emission at 477 and 651 nm was used for the selective detection of Pb2+ and pyrophosphate (PPi) with LODs as low as 0.055 mmol/L and 0.089 mmol/L, respectively [197]. Fu’s team [198] has introduced a μPad integrated with a portable fluorometric system for formaldehyde (CH2O) detection in the linear range of 0.2–2.5 ppm.

Fluorescent paper sensors also have been applied for monitoring various chemicals like antibiotics [199-201] and pesticides [202-204]. μPads were designed for the detection of tetracycline (TC), relying on multi-color fluorescence change of a glove-based visual probe [199]. Combined with smartphone-based chromaticity analysis APP, the portable detection of TC was obtained with LOD of 9.5 nmol/L (Fig. 4e). Jiang’s group [203] has reported a dual-emissive ratiometric paper strip consisting of an UV lamp and 3D-printing technology for the smartphone-based analysis of pesticide in a “on–off-on” fluorescent mode with a LOD of 59 nmol/L (Fig. 4f).

Surface-Enhanced Raman Scattering (SERS)

SERS sensors on paper have been a hot detection method in recent years. When the nanomaterials are modified on the paper surface, SERS can be increased by the nanomaterials. For example, the gold/silver nanoparticles (Au/AgNPs) drop on the paper. Then it will produce precipitation and generate hot spots to increase the sensitivity of detection. SERS-based test samples can be divided into three categories.

Environmental Applications

The exploration of content of rhodamine (R6G) in rain water had been operated successfully by constructing a 3D SERS paper strip. R6G can be selectively detected with the minimum magnitude of 1 × 10–11 mol/L by using silver mirror reaction [205]. Also, Au/AgNP-based paper sensor was used to explore rhodamine B (RhB) and crystal violet (CV) in deionized water and tap water [206-208]. Kim’s team fabricated the M13 bacteriophage-functionalized silver nanowires (AgNWs) SERS sensor for capturing pesticides, especially paraquat (PQ) [209]. Zhang’s group has developed the 3D Silver Dendrites for the determination of Neonicotinoid with the LOD of 0.02811 ng/mL. The platform made great contributions for detecting various contaminants [210]. In 2021, Liu’s team prepared an MXene (Ti3C2Tx) -Ag nanoparticles (NPs) hybrid SERS biosensor for detecting adenine molecules in biological environment with the LOD of 1 × 10−8 mol/L [211]. Wang’s group used superhydrophobic SERS substrates to detect nitenpyram in the field of agriculture with the LOD of 1 nmol/L [212]. Some applications in environment [213-216] are shown in Table 3.

Table 3

Summary of the corresponding SERS applications on µPad in the fields of the environment, food, biochemistry in 2021

Materials	Operation method	Target molecule	LOD	Samples	Refs.
A mixture of silver nanoparticle (AgNP) and gold nanostar (AuNS)	Dropping the solution on cellulose nanofiber (CNF)	Ferbam	50 μg/kg	Kale leaves	[213]
Colloidal nanoparticles	Spraying nanoparticles onto hydroxyethyl cellulose (HEC)	Thiram	1 × 10–7 mol/L	Mud	[214]
Silver nanodots on three-dimensional cellulose fibers	A magnetic bead-based separation method	R6G (II), TAMRA	153.53 and 230.37 pmol/L for R6G and TAMRA	Dyes	[215]
Au@Ag core-shells	A electrospun paper matrix	methamphetamine	7.2 ppt	Wastewater	[216]
Silver nanoparticles	Immersing nanoparticles in melted wax vessel	2,4-dichlorophenoxyacetic acid	1.0 × 10–4 mg/g	Green tea	[247]
Au@tannic acid (TNA) substrate	In-situ growth on paper	Reductants	–		[248]
4-MBA-functionalized Au@ZIF-8 SERS paper	Plasma reduction method	Putrescine and cadaverine	76.99 and 115.88 ppb	Spoiled salmon, chicken, beef, and pork samples	[249]
Nanogold particles	Dropping nanoparticles on plasma-printed substrate	Cocaine	1 ng/mL	Cocaine	[250]
Uniform gold nanospheres treated by chloride ion	Self-assembling nanoparticles on paper	Fentanyl citrate	0.59 μg/mL and 2.78 μg/ mL	Urine and serum	[268]
Gold nano-pyramid arrays	Dropping nanoparticles on paper	S-100β	5.0 pg/mL	Blood plasma	[269]
Gold nanostar@Raman reporter@silicasandwiched nanoparticles	Dropping nanoparticles on paper	Carcinoembryonic antigen	1.0 ng/mL	Whole blood	[270]
Gold nanoparticles (Au NPs)	Dropping nanoparticles	Serum	10 ppm	Blood	[271]
Silver-nanowires	Dropping nanoparticles on paper	DNA	3.12 pg/μL	Various bacteria and viruses	[272]

Food Applications

In Poppi’s group, AuNP-based paper substrate was applied in SERS to detect crystal violet sample and to explore the amount of nicotine and uric acid. The respective LODs were 20 μg/L for nicotine and 30 μg/L for uric acid [217]. In order to get a sensitive SERS signal, AgNPs/RGO, AgNF/AgNP arrays, AgNP and AuNP based paper substrates were also used in the field of food applications [218-221]. Pesticides were detected by paper-based SERS method [222]. Silver nanoparticles and graphene oxide were printed on the paper surface. Thiram, thiabendazole and methyl parathion were measured with low LOD. A two-dimensional MoO3-x nanosheet ink was produced in Lan’s group to test crystal violet and malachite green on the fish surface by office inkjet printer [223]. Rhodamine 6G and rhodamine B can be detected with SERS in vegetables and contaminants in rain, pond, and tap water [224]. A sensitive SERS detection of R6G with a linear range of 1 × 10−9–1 × 10−5 mol/L and a detection limit of 1 × 10−11 mol/L was also realized [225]. Xu’s team [226] used SERS signal intensity and chiral signal intensity to detect different concentrations of C. jejuni spiked in milk samples with a good linearity from 1 × 102 to 1 × 106 cfu/mL. Also, Zhang’s group has detected melamine in the sample of milk based on paper SERS with a LOD of 1 ppm and a good linear correlation (1–1000 ppm) [227]. Haddad’s group provided a simple and sensitive way for analysis of fentanyl in Heroin [228], and Li’s group has detected SO2 in wine from 1 μmol/L to 2000 mmol/L with μPads SERS sensor [229] (Fig. 5a). Besides, it is very important to detect drug concentration. Ameku’s group [230] has designed a μPAD based SERS to identify caffeine, paracetamol, and levamisole adulterants simultaneously. In our daily life, it is important to detect some dyes’ concentration in food safety field. Gu’s group presented a novel seed-mediated growth method for making a SERS device. The method can detect Methylene Blue with LOD down to 1 × 10–9 mol/L. Also, the LOD was 1 × 10–8 mol/L for both Crystal Violet and Rhodamine 6G solution [231]. AgNF based paper-SERS [232] and pressure paper spray mass spectrometry (PS-MS) were all used to detect ketoprofen with LODs of 0.023 and 0.076 mg/L respectively. SiO2/Ag nanocomposite-based paper substrate [233] was applied to detect acrylamide (AAm) with SERS (Fig. 5c). Dao’s group [234] has developed a new detection method for monitoring the pesticide chlorpyrifos with paper SERS. Lv’s group found that MoS2@Au/Ag hybrid- based paper device exhibited a distinct advantage to separate and preconcentrate in biological and chemical detection [235] (Fig. 5b). Chen’s team has fabricated µPAD combined with SERS for exploring sulphite in wine, which showed a good linearity from 5 to 300 μg/mL [236]. Huang’s group explored a novel label-free 3D-SERS substrate with black phosphorus-Au filter paper, which can detect three types of target bacteria including Staphylococcus aureus, Listeria monocytogenes and Escherichia coli at the same time [237] (Fig. 5d). Paper-based lateral flow immunoassay (LFIA) based on (Fe3O4/Au-PEI) nanoparticles tested bacteria in urine and milk samples with a good linearity from 1 × 101 to 1 × 107 cfu/mL in less than 60 min [238]. A paper-based SERS sensor with AgNPs can detect methyl parathion quickly in the sample of fruit [239], tartrazine in Children's snacks [240], crystal violet (CV) and the fungicide thiram in food [241]. Wu’s team has separated and identified lycopene and β-carotene in food products successfully with paper SERS [242]. Cellulose nanofibers (CNF)/AuNP nanocomposite-based paper SERS sensor was used to detect thiram with the LOD of 52 ppb [243]. Also, SERS sensor showed a LOD of 1 × 10–7 mol/Lfor methylene blue in the sample of apples [244]. Ag@SiO2 core–shell nanoparticles [245] were used on filter paper to fabricate SERS chips. The chips were used for detecting the amount of thiram with the LOD of 1 × 10–9 mol/L, which had great potential in pesticide residues’ detection. Yang’s group has explored SERS chips with Ag/Au NPs to detect malachite green, methylene blue and crystal violet with LODs of 4.3 × 10−9, 2.0 × 10−8, and 8.1 × 10−8 mol/L, respectively. [246]. Other applications in food [247-250] are shown in Table 3.

Fig. 5

Some examples for SERS detection. a Schematic illustration of dual-modal detection of SO2 [229]; b Schematic illustration of fabrication of MoS2@Au/Ag hybrid substrate for SERS [235]; c Schematic illustration of F-SANC substrate fabrication in SERS detection of AAm [233]; d Schematic illustration of BP-Au filter paper-based SERS substrates for food analysis [237]; e Schematic illustration of SERS paper-based lateral flow strip (PLFS) [252]; f Schematic illustrations of paper-based SERS for serum bilirubin detection [266]

Biological Applications

SERS based paper has also been used in the biological applications. Paper substrate and its biosensing application such as picomolar SERS based paper was used to detect folic acid in picomolar scope for healthcare [251]. SERS paper-based lateral flow strip (PLFS) was good for assisting screening of traumatic brain injury (TBI) patients in a short time. It was used to detect neuron-specific enolase (NSE) with a LOD of 0.86 ng/mL [252] (Fig. 5e). Qi’s group has reported that by using DNA-encoded Raman-active anisotropic nanoparticles on paper, microRNA can be sensitively detected within 15 min with the LOD of 1 pmol/L [253]. SERS can also be used for distinguishing Zika and dengue nonstructural protein 1(NS1) biomarkers with a high sensitivity [254]. Lorenzo Russo has adopted paper-based immunoassays by SERS with AuAg nanoshells for detecting the biomarker of resistance protein A (MxA) [255]. What’s more, a dipstick immunoassay was realized by using AuAg NSs conjugated antibody as a “nanotag”. For biomarker detection, SERS-based µPAD can be used for quantitative detection of multiple cardiac biomarkers simultaneously. Glycogen phosphorylase isoenzyme BB (GPBB), Troponin T (cTnT) and CK-MB for early diagnosis have been explored simultaneously [256]. SERS containing graphene-isolated-Au-nanocrystals was used to detect bilirubin [257]. Different sampling methods have also been realized in SERS. For example, Merve Eryılmaz has explored SERS-based lateral flow immunoassay (LFIA) test strips for Group A Streptococcus pyogenes (GAS) detection. By using of the swab sampling technique, SERS-based rapid assay got the LOD of 0.2 CFU/mL for GAS [258]. Also, nanopaper-based analytical devices (nanoPads) were appeared for a new platform. The devices were the natural hydrophilicity and hollow-channel. Pump-free can be realized on the nanopaper. SERS can be performed on this new platform for environmental pollutants detection [259]. A gold-coated magnetic nanoparticle with anti Microcystin-LR (MC-LR) antibody Fab fragments was produced. The relavent antigen can be recognized from aqueous media and blood plasma [260]. In clinical analysis field, disease-related substances are important and SERS has successfully realized the disease detection. A paper-based SERS assay was used to explore atherosclerosisa [261]. A nanoporous networking membrane was adopted as the substrate and SERS nanotags was used as the signal reading probe. And the LOD was 0.1 pg/mL. Magnetic separation was realized by plasmonic paper-based SERS and the capture accuracy of the HT-29 cells was 83.7% [262]. Lu has reported the simultaneous detection of two biomarkers of squamous cell carcinoma antigen (SCCA) and osteopontin (OPN) by paper-based SERS method. Au–Ag nanoshuttles (Au-AgNSs) was used as SERS tags. Au nanoflowers (AuNFs) were used to develop a sandwich structure for later immunoassay. The LODs for SCCA and OPN in human serum were 8.628 pg/mL and 4.388 pg/mL, respectively [263]. Zavyalova’s group developed a paper-based device for detection of viruses with SERS. SARS-CoV-2 virus can be measured rapidly with better selevtivity [264]. In 2017, a plasmonic filter was used by Wang’s group to filtrate, capture and identify Streptomycete spores with SERS method. The device can discriminate the source of nosiheptide product. With this filter, a stain- and PCR-free detection was realized with only 5 μL sample solution and 5 min for the detection time [265]. As reported by Pan [266], a paper-based SERS biosensor was established for free bilirubin detection by label free method. Enrichment function was coupled on this sensor and multifunctional graphene oxide-plasmonic gold nanostar (GO-GNS) hybrids was adopted (Fig. 5f). Adenosine can be used for cancer biomarker. A SERS-chemometric method was established for the detection of urinary adenosine [267]. Some applications in biological [268-272] in 2021 were shown in Table 2.

Chemiluminescence (CL)

To perform sensitively and rapidly in POCT, paper-based platforms in CL system arises great concern.

A novel paper-based CL system with H2O2-rhodamine b (RhoB) and MOF was established. MOF used Co2+ as the central ion. The CL system was used for total phenolic content detection. The LODs for gallic acid, quercetin, catechin, kaempferol and caffeic acid were 0.98, 1.36, 1.48, 1.81 and 2.55 ng/mL, respectively [273] (Fig. 6a). Yahyai reported that polyphosphate (PP) can enhance the CL of graphene quantum dots (GQDs)-KMnO4 system. Deltamethrin (DM) can quench this system’s CL. The mechanism was discussed and the CL luminous body was Mn2+. DM can be detected in food samples with the LOD of 0.15 μg/mL [274]. Montali et al. [275] presented a CL foldable paper-based biosensor based on three coupled enzymatic reactions catalyzed by enzyme acetylcholinesterase (AChE), choline oxidase and horseradish peroxidase. The enzyme can catalyze the decomposition of hydrogen peroxide and then organophosphorus (OP) can be detected with its inhibiting effect of AChE. In Yang’s work, Co2+/N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co2+-ABEI-Fe3O4@void@C) was used on a three-dimensional microfluidic paper-based device (3D μPAD) to detect early acute myocardial infarction (AMI) biomarkers in human serum samples. The time-resolved CL signals were used for the simultaneous determination of AMI biomarkers [276] (Fig. 6b). As reported by Li, temporal resolution CL method can be performed with double-layered 3D μPAD. Then glucose, lactate, cholesterol and choline can be detected at the same time. H2O2 was produced after the reaction of enzyme and substrate. The luminol-H2O2 CL system was still catalyzed by the cobalt ion. With temporal resolution CL method, the LOD for glucose, lactate, cholesterol and choline was 8, 15, 6 and 0.07 μmol/L, respectively [277] (Fig. 6c). In the future, paper-based CL immunodevice by controlling reagent transport can provide a new way of sensitive detection of multi-biomarkers in a short time. For bioanalysis, in Han’s work, combined with enzyme-catalyzed CL method, the testing of cardiac troponin I (cTnI) in human serum samples with LOD of 0.84 pg/mL was achieved [278].

Fig. 6

Some examples for CL detection. a Schematic illustration of μPAD for phenolic compounds detection [273]; b Schematic illustration of 3D μPAD for copeptin, h-FABP and cTnI detection [276]; c Illustration for multiplexed CL analysis on 3D μPAD. [277]; d Schematic illustration for the mechanism of plasma treatment of paper for antibody immobilization [15]

In recent years, our lab has worked on paper-based CL sensing platforms. The paper-based chip was used in biomedical and environmental fields. For instance, a wax-printed CL μPad for the ofloxacin detection was shown, combined with the luminol-H2O2-OFLX system enhanced by AgNPs was developed. The LOD for OFLX was 3.0 × 10–10 g/mL [279]. A molecularly imprinted polymer (MIP) was successfully synthesized on the paper surface for the CL detection of dichlorvos (DDV). The LOD for DDV detection was 0.8 ng/mL [280]. A paper-based CL immunodevice prepared by a low-cost antibody immobilization method based on plasma treatment was introduced. The detection of CEA in human serum was performed with a linear range from 0.1 to 80.0 ng/mL [15] (Fig. 6d). In 2017, a paper-based CL immunodevice by using controlling reagent flowing technique was explored. The technique can change the migration rate for the reagent and then the time-dissolved CL detection can be realized on the paper-base device. CEA, carcinoma antigen 125 (CA125) and carbohydrate antigen 199 (CA199) can be detected simultaneously on the paper-based chip with LODs of 0.03 ng/mL, 0.2 U/mL and 0.2 U/mL, respectively [281]. In 2018, carbon nanospheres with HRP functionalization were used as signal antibody markers to construct a paper-based CL immunodevice for the determination of CEA with the LOD of 3 pg/mL. The method was nearly 10 times more sensitive than commercial Ab2-HRP kits [282]. In 2019, a 3D washing strategy was developed on a paper-based immunodevice using a ring-oven. The 3D washing strategy had a lower background than the flat washing mode, because non-specific binding proteins could be continuously transported to the waste zone by gravity and capillarity. A low LOD of 2 pg/mL was obtained for the detection of CEA by CL [283]. In 2019, a new fabrication method was used to manufacture a μPad. The recycled polystyrene in chloroform was used as a hydrophobic reagent. A tape mask was adopted to protect the hydrophilic channel. Three cancer biomarkers, CEA, α-fetal protein (AFP), prostate-specific antigen (PSA) in human serum samples on the μPAD were detected by luminol-H2O2 p-iodiophenol (PIP) CL system. The linear ranges were 0.05–80.0 ng/mL, 5.0–80.0 ng/mL, 1.0–50.0 ng/mL, respectively [284]. PSA was detected sensitively on a μPAD [285] by using NH2-MIL-53(Fe) as the detection antibody label. The dual mode detection (FL and CL) was achieved with the LODs of 0.3 ng/mL for CL and 0.2 ng/mL for FL. In 2021, it was reported that by using Co-Fe Prussian blue analogue nanocubes (Co-Fe PBA NCs), the strong CL still happened in the absence of H2O2 on a paper-based CL device [286].

Conclusion and Outlook

µPads have been widely used for inorganic ions, organic compounds, proteins, nucleic acid and drug analysis due to the advantages of low-cost, easy-to-fabrication, strong-capilary action and biological compatibility. From the perspective of material synthesis substrate, by in-situ growth on paper chip or in-situ dropping on paper, it can realize detection more sensitively and faster. From the perspective of paper chip design, different injection areas or reaction areas are designed on the surface of the paper base to build a paper-based platform with diversified functions, which can satisfy the requirement of rapid detection of single component or multi-component samples. At present, the commonly used paper-based detection methods are mainly EC, ECL, colorimetry, FL, SERS and CL.

EC method is attractive alternative detection technique for μPads because of its portable size, small instrumentation and high sensitivity. However, the stability of detection electrodes, which corresponds to temperature, pH and the fabrication cost, still remain a challenge.

Due to the high sensitivity and signal-to-noise ratio of ECL analysis, low detection limits have been achieved for miRNA, tumor cell MCF-7, heavy metal ions, antigens and streptavidin since 2015. The development of equipments is limited, which requires the continuous efforts of scientists. Colorimetric methods have become the most frequently used ones in µPads because the signal readout method is simple. Distance-based and lateral flow assay paper analytical devices are well-established platform because of easy integration with POCT devices. FL detection is a highly sensitive and selective optical analysis technology that can be used for different fields. Paper-based SERS sensors have the advantages of low cost and simple sample collection, but the hydrophilic surface inhibits its sensitivity. This can be improved by modifying nanomaterials on the surface. Then paper-based SERS sensors can be used for the analysis of environmental samples, food samples and biological samples. CL analysis is sensitive and fast. The paper-based CL immunoassay devices have the characteristics of controlled reagent delivery, which provide strategies for detecting various antigens and biomarkers of early cancer.

Although paper-based platform has been widely used in various fields, sample pretreatment is still needed in most cases. It still needs a lot of efforts to build paper-based platform to test actual samples directly. In addition, another challenge is that the paper-based devices are not connected to the common products in our daily life, so it is exciting to realize simpler and faster detection mode and build a life-experiment integrated platform. Finally, the construction of paper laboratory is also a promising platform. We are looking forward for more designs to indeed realize the micro total analysis on μPads. In addition, POCT has the outlook for home-stay diagnosis on the paper chip; we always believe that more and more people will build multi-dimensional platforms through paper for home-stay diagnosis. Through our joint efforts, paper-based platforms will play an infinite possibility in the future.