The robust identification and quantification of various biomarkers is of utmost significance in clinical diagnostics and precision medicine. Fluorescent immunoassays are widely used and considered as a gold standard for biomarker detection due to their high specificity and accuracy. However, current commercial immunoassay tests suffer from limited detection sensitivity and complicated, labor-intensive operation procedures, making them impractical for point-of-care diagnosis, particularly in resource-limited regions. Recently, microfluidic immunoassay devices integrated with plasmonic nanostructures have emerged as a powerful tool for sensitive detection of biomarkers, addressing specific issues, such as integration schemes, easy operation, multiplexed detection, and sensitivity enhancement. In this paper, we provide a discussion on the recent advances in the plasmonic nanostructures integrated with microfluidic devices for fluorescent immunoassays. We shed light on the nanofabrication strategies and various fluidic designs for rapid, sensitive, and highly efficient sensing of antigens. Finally, we share our perspectives on the potential directions of these integrated devices for practical applications.

The ability to detect and quantify target antigens is highly demanded for clinical diagnostics and personalized medicine. Currently, the “gold standard” for biomarker detection is based on enzyme-linked immunosorbent assays (ELISA), which utilize antibodies conjugated with enzymes for signal amplification.1 Yet, conventional ELISA involves burdensome operation steps, skilled personnel, and costly equipment for enzyme reaction and fluorescence measurement. In particular, samples need to be transported to professional laboratories for further processing with high-cost kits and instruments, which causes delays or detection lags up to several hours between sample collection and ultimate results. Thus, it is not suitable for on-site screening or point-of-care diagnosis, especially for some acute or infectious diseases.

The advent of lab-on-chip devices has revolutionized conventional immunoassays with the merits of miniaturized apparatus, sample economy, compactness and portability, high throughput, and the capability to multiplex and automate.2,3 A common feature of such devices is that they integrate various operational procedures, including sampling, mixing, reaction, and readout, into single and small microfluidic chips to automatically complete the whole process for rapid and intuitive assay results. This technology delivers a practical handheld tool for simple, reliable, fast, and inexpensive near-patient testing of multiple biomarkers, thereby greatly reducing medical expenses and increasing patients' access to resource-limited settings.4 The principle of downscaling, however, brings up the issue of quantifying target biomarkers in extremely low amounts of samples at low concentrations. With regard to the microfluidic assay platforms, they face up with the challenges of achieving high sensitivity due to the low quantum efficiency, photobleaching, and autofluorescence. The detection limit of reported works is usually in the range of 1 ng/ml down to 100 pg/ml with functionalized poly(dimethylsiloxane) (PDMS) or glass microchannels,5 which fails to satisfy the requirement of trace biomarkers in saliva or serum. Thus, significantly improving the sensitivity of microfluidic assay devices is still in urgent demand for early disease diagnosis.

Various effective strategies have been developed to increase the sensitivity of these microfluidic assay devices.6 Existing techniques, including mass spectrometry,7 electrochemical analysis,8 magnetic beads,9 and plasmonic nanostructures, have demonstrated significant sensitivity enhancement in biosensing, as listed in Table I. Mass spectrometry, though exhibiting excellent sensitivity in sensing applications, suffers from bulky equipment and skilled personnel. Electrochemical methods provide an effective way for inexpensive and rapid biomarker analysis. Progresses are still needed in reducing sample volume and multiplexed sensing. Magnetic bead-based assays may encounter the issue of non-specific binding, resulting in a low signal-to-noise ratio (SNR). In contrast, the incorporation of nanostructures into microfluidic assay platforms offers a simple, fast, and handheld platform that does not require expensive equipment or complicated operation steps, yet maintaining high sensitivity and accuracy for on-site disease screening.10–14 Categorized by the sensing mechanisms, the methods based on plasmonic nanostructures include Surface Plasmon Resonance (SPR),15 Localized Surface Plasmon Resonance (LSPR),16 Surface-Enhanced Raman Scattering (SERS),17–19 and Metal-Enhanced Fluorescence (MEF).20 SPR and LSPR rely on refractive index and show good performance in biosensing. However, the involved optical systems can be hardly integrated. SERS has ultra-high sensitivity but still requires bulk and expensive Raman spectrometer.21 MEF is a fluorescent-based method that is widely used and poses great potential for future point-of-care diagnostics.

TABLE I.

Comparisons of biosensing techniques for microfluidic assays.

TechniqueSensing mechanismCostAnalytesLimit of detectionReference
Mass spectrometry Mass-to-charge ratio High Lysine 30 fM (0.5 ng/ml) 7  
Electrochemistry Electrical Moderate Lidocaine 1 μ8  
Magnetic beads Fluorescence Low cTnI 9.56 pg/ml 9  
hFABP 92.5 pg/ml 
NT-proBNP 5.29 pg/ml 
SPR Refractive index Moderate IgG 1.3 nM (195 ng/ml) 15  
IgM 2.4 nM (2280 ng/ml) 
LSPR Refractive index Moderate CEA 0.4 ng/ml 16  
SERS Characteristic peaks High TP53 2.26 aM 17  
PIK3CA-Q546K 2.34 aM 
MEF Fluorescence Moderate Dopamine 0.21 nM 20  
TechniqueSensing mechanismCostAnalytesLimit of detectionReference
Mass spectrometry Mass-to-charge ratio High Lysine 30 fM (0.5 ng/ml) 7  
Electrochemistry Electrical Moderate Lidocaine 1 μ8  
Magnetic beads Fluorescence Low cTnI 9.56 pg/ml 9  
hFABP 92.5 pg/ml 
NT-proBNP 5.29 pg/ml 
SPR Refractive index Moderate IgG 1.3 nM (195 ng/ml) 15  
IgM 2.4 nM (2280 ng/ml) 
LSPR Refractive index Moderate CEA 0.4 ng/ml 16  
SERS Characteristic peaks High TP53 2.26 aM 17  
PIK3CA-Q546K 2.34 aM 
MEF Fluorescence Moderate Dopamine 0.21 nM 20  

This Perspective aims to provide an explorative review on microfluidic devices using MEF for sensitive fluorescent assays. First, we briefly introduce the fundamental principles of metal-enhanced fluorescence based on plasmonic nanostructures. Subsequently, an overview of existing fabrication techniques that integrate nanostructures into microfluidic chips will be reviewed. Next, we highlight several key paradigms of microfluidic assay devices that employ integrated plasmonic structures for fast and sensitive quantification of biomarkers (Fig. 1). Finally, we provide our perspectives on the potential directions and technical innovations for applying these devices into real-life applications.

FIG. 1.

Schematic illustration of microfluidic assay devices integrated with plasmonic nanostructures.

FIG. 1.

Schematic illustration of microfluidic assay devices integrated with plasmonic nanostructures.

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Metal-enhanced fluorescence refers to the phenomenon that the fluorescent intensity would greatly increase when fluorophores are positioned in the near-field of plasmonic metal nanostructures. This is mainly due to the LSPR generated by collective oscillations of free electrons around metallic surfaces, which enhances the electromagnetic field nearby.22–24 

Although the underlying mechanism of metal-enhanced fluorescence is still controversial, the influencing factors mainly include the increase in excitation rate (absorption) and quantum yield. Therefore, this effect can be mainly attributed to two reasons. One is the enhancement of excitation efficiency. The presence of metal nanostructures and the associated localized surface plasmon resonance enhance the excitation rate of fluorophores by opening additional electron configurations. The other is the increase of the radiative decay rate Γ, which determines the quantum yield Q 0 and lifetime of the fluorophore ( τ 0 ) by10,25–27
Q 0 = Γ Γ + k nr ,
(1)
τ 0 = 1 Γ + k n r ,
(2)
where k nr refers to the non-radiative rate. According to Eq. (1), the increase of radiative decay rate Γ raises the quantum yield Q 0 and results in brighter fluorescence intensity. Meanwhile, the fluorescence lifetime becomes shorter as shown in Eq. (2), which improves the photostability. In addition, non-radiative energy transfer occurs during the emission process from the metallic surfaces to fluorescent molecules. This new energy pathway strengthens the emission of fluorophores to the far-field and, therefore, enhances the intensity. It should be noted that the electric field exponentially decays away from the nanostructures.17 Thus, the enhancement normally happens within the distance between a few nanometers to tens of nanometers. As the fluorophores move closer to the surfaces (<5 nm), the fluorescence would be quenched by the metal. It is widely accepted that the best quantum yield and photostability can be achieved when the separation distance falls in the ranges of 5–90 nm, depending on the morphology of plasmonic structures.12 

The morphology of plasmonic nanostructures, including materials, shapes, sizes, and pitches, plays a crucial role in determining the fluorescence enhancement factor. The reported plasmonic nanostructures range from island films, nanoparticles, nanocolloids, and various nanoarrays, usually made of metallic materials. The fabrication and integration of these nanostructures can be conveniently categorized into bottom-up and top-down strategies.

The solution-based bottom-up technique is frequently applied to prepare metallic nanoparticles, which features a simple procedure of wet chemical reactions. The most commonly used protocol proposed by Lee and Meisel is leveraged on the citrate reduction for both silver and gold nanoparticles.28 These nanoparticles can be attached on appropriate substrates and the hot spots between the gaps of them generate surface plasmons for MEF. The morphology and aggregation features of these nanoparticles could affect the maximum field strength and, hence, the enhancement factor of fluorescence. A number of researchers have reported various nanoparticles with different shapes and sizes by unique solution reactions, including nanospheres,29–31 nanorods,32–34 nanotriangles,35–37 and nanowires.38–40, Table II summarizes representative achievements using solution-based nanoparticles for fluorescence enhancement. As listed, the enhancement factor was relatively limited at below 100 and the reproducibility is compromised due to the random distribution of these nanoparticles. It is challenging to accurately control the chemical process to obtain uniform particle size, morphology, and crystal structure. The incorporation of these plasmonic particles into microfluidic chips for bioassays requires cumbersome preparation steps with issues of reproducibility, stability, and uniformity. Moreover, these techniques are not compatible with semiconductor microfabrication, thus not suitable for large-scale production and applications. An improved alternative is the seed-mediated growth mechanism, which employs metallic seeds coated on the substrates for controlled growth of nanostructures.41,42 With a high monodispersity and homogeneous morphology, the enhancement factor can be increased to several hundred for highly sensitive assays.

TABLE II.

Typical examples of nanoparticles for fluorescent enhancement using solution-based synthesis methods.

AuthorsNanoparticleCompositionSynthesisEF
Sugawa et al.29  Nanospheres Copper coated on silica/130 nm Stöber method (NH3, TEOS) 89.2 
Ren et al.32  Nanorods Au and Ag nanorods HAuCl4, AgNO3, CTAB 14.5 
Aslan et al.35  Nanotriangle Ag nanotriangle/100 nm AgNO3, CTAB 16 
Abel et al.38  Nanowires Ag nanowires/72 nm AgNO3, PVP, EG 2.2 
AuthorsNanoparticleCompositionSynthesisEF
Sugawa et al.29  Nanospheres Copper coated on silica/130 nm Stöber method (NH3, TEOS) 89.2 
Ren et al.32  Nanorods Au and Ag nanorods HAuCl4, AgNO3, CTAB 14.5 
Aslan et al.35  Nanotriangle Ag nanotriangle/100 nm AgNO3, CTAB 16 
Abel et al.38  Nanowires Ag nanowires/72 nm AgNO3, PVP, EG 2.2 

Top-down schemes involve conventional cleanroom-based microfabrication techniques to directly transfer artificial patterns onto substrates for desired plasmonic arrays. Typical top-down routines include electron-beam lithography (EBL) and focused ion beam (FIB) lithography, which suffer from expensive equipment and low throughput. Nanoimprint can be an alternative that uses the mold to form large-area nanostructures. Leveraging this technique, Chou's group demonstrated three-dimensional plasmonic nano-antenna-dots with a remarkably high fluorescence enhancement factor of over 7400-fold.43 Nevertheless, the preparation of imprint template is cumbersome and during the process, defects could be introduced due to the uneven pressure and mold deformation. Colloidal lithography has been also reported for large-scale production of plasmonic patterns with self-assembled colloid arrays and subsequent etching.44,45 The monodispersion of well-ordered colloidal arrays provides lithographic masks for patterning on the substrate. More importantly, the modification of self-assembled colloidal masks offers possibilities for creating various plasmonic nanopatterns with different sizes and shapes. In colloidal lithography, however, defects may be possibly introduced during the self-assembly of spheres. Studies have been devoted to developing defect-free colloidal arrays over a large area using pre-patterned templates or interfacial precursors. The compatibility with conventional lithography is also explored to combine “bottom-up” self-assembly with “top-down” patterning and etching for working devices.

Another interesting nanopatterning technique is glancing angle deposition (GLAD), a physical vapor deposition method for the fabrication of thin films with engineered nanostructures.46–49 It utilizes the so-called shadow effect, which means the initial nucleation of atoms favors the further growth of nanocolumns during evaporation. The morphology of the patterned film can be tailored through the substrate motion, including rotation and tilt angle. GLAD is based on the one-step deposition commonly used in microfabrication, eliminating the requirement of complex preparations and costly tools. The utility of these structures for plasmonic fluorescence enhancement is verified by several groups, with the reported enhancement factors in the range of several hundred.50,51 This easy protocol provides a potential method for the fabrication and integration of nanostructures.

The immunosensors for precise measurement of biomarkers usually exploit fluorescence detection as an effective biorecognizer. Fluorophore labels provide special affinity with target biomolecules and act as excellent signaling probes for sensitive and quantitative molecular sensing through the measurement of intrinsic properties, including fluorescent intensity or wavelength shift. Due to the merits of simple operation, fast response, and high versatility, fluorescent immunosensors have become one of the most developed and promising biological detection technologies in biosensor applications. Still, some issues remain to be addressed. One of the critical issues for their practical applications is the sensitivity or detection limit, especially for the quantification of low-abundant biomarkers. For example, in the early diagnosis of some diseases, the concentration of target analytes is too low to be detected by conventional optical apparatus. Other limitations include a low SNR caused by non-specific binding and interference from spontaneous fluorescence, tedious operation procedures including fluorescent labeling and repetitive washing steps as well as low efficiency for multi-target detection. Consequently, ultrasensitive biosensors with the characteristics of low cost, easy operation, and high efficiency are potentially required for analytical applications in point-of-care diagnosis. The incorporation of MEF into biosensing amplifies the fluorescent signals and, thus, lowers the limit of detection (LoD) with high sensitivity. To date, microfluidic immunoassay platforms integrating with plasmonic nanostructures have shown substantial values and notable development in three directions: (1) multiplexed and parallel detection for various biomarkers, (2) ultrasensitive detection capability, and (3) one-step immunoassays. Some representative works are summarized in Table III and discussed in detail below.

TABLE III.

Microfluidic assay devices integrated with plasmonic nanostructures.

AuthorsCharacteristicsNanostructuresBiomarkersLoDDetection time
Yu et al.52  Multiplexed and parallel detection ZnO nanorods H2N5, H7N2 3.6 × 103 EID50/l 1.5 h 
Chen et al.53  Au nanorods Serum cytokine (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-r) 5 pg/ml 40 min 
Kannegulla et al.54  Ultrasensitive detection Open-ring nanoarrays DNA ∼300 fM … 
Zhao et al.55  Zn(OH)F arrays HE4 protein 9.3 fM … 
Jalali et al.56  Au nano/microisland E. coli, MRSA 50 CFU/ml … 
Hao et al.57  Ag nanoparticle-deposited ZnO nanorod arrays Exosome 20 exosomes/μ… 
Kim et al.58  Silica-coated silver nanoparticles AChE 10−10 M … 
Shen et al.59  Au nanoclusters H2O2 200 attomole/cell … 
Xu et al.60  Gold islands AFP 94.3 fg/ml … 
Wang et al.61  Silver nanoparticles miRNA 10−9 M … 
Kim et al.62  One-step immunoassays Gold particles (GNPs) amyloid β () and Tau protein 170 and 190 fM >40 min 
Hong et al.63  Mesoporous silica-coated gold nanorods IVA 0.52 pg/ml <20 min 
Wang et al.64  Au nanorods cTnI ∼1.4 fM <6 min 
AuthorsCharacteristicsNanostructuresBiomarkersLoDDetection time
Yu et al.52  Multiplexed and parallel detection ZnO nanorods H2N5, H7N2 3.6 × 103 EID50/l 1.5 h 
Chen et al.53  Au nanorods Serum cytokine (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-r) 5 pg/ml 40 min 
Kannegulla et al.54  Ultrasensitive detection Open-ring nanoarrays DNA ∼300 fM … 
Zhao et al.55  Zn(OH)F arrays HE4 protein 9.3 fM … 
Jalali et al.56  Au nano/microisland E. coli, MRSA 50 CFU/ml … 
Hao et al.57  Ag nanoparticle-deposited ZnO nanorod arrays Exosome 20 exosomes/μ… 
Kim et al.58  Silica-coated silver nanoparticles AChE 10−10 M … 
Shen et al.59  Au nanoclusters H2O2 200 attomole/cell … 
Xu et al.60  Gold islands AFP 94.3 fg/ml … 
Wang et al.61  Silver nanoparticles miRNA 10−9 M … 
Kim et al.62  One-step immunoassays Gold particles (GNPs) amyloid β () and Tau protein 170 and 190 fM >40 min 
Hong et al.63  Mesoporous silica-coated gold nanorods IVA 0.52 pg/ml <20 min 
Wang et al.64  Au nanorods cTnI ∼1.4 fM <6 min 

Biomarkers contained in blood, urine, saliva, and other body fluids are biological indicators that are used to predict a clinically relevant process. However, a single protein biomarker may not be highly specific for a target disease, making the detection of a single target insufficient and inaccurate for diagnosis. Simultaneous analysis of multiple biomarkers in a single assay provides richer and more scientifically accurate information for comprehensive diagnosis of a biological sample.13 The most straightforward way for multiplexed detection is the parallel arrangement of microfluidic channels for individual assays using a panel of capture antibodies.65,66 Typical examples include the ZnO or Au nanorods integrated microchip reported by Yu et al. and Chen et al., respectively. Both utilized different branched microchannels and spatial encoding of capture antibodies for multiplexed detection [Fig. 2(a)].52,53 Benefiting from microfabrication techniques, these devices can be easily scaled down for more biomarkers. Another notable scheme to implement multiplexing is the utilization of a bead-based protein capture method to label and detect various protein targets on beads.67 These bead-based assays eliminate the requirement of washing steps and can be analyzed through flow cytometry. Alternatively, protein microarrays are established where capture antibodies are immobilized onto planar surfaces in periodic arrays for highly miniaturized and parallelized assays. Such assay systems consume only tiny amounts of sample and reagent volumes. Through multiplexing, a panel of proteins, pathogens, bacteria, and DNA/RNA can be detected simultaneously, which provides a basis for early diagnosis of diseases.

FIG. 2.

(a) Portable devices for the detection of multiple pathogens. Reprinted with permission from Yu et al., Small 13, 1700425 (2017). Copyright 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) The investigation of the fluorescence enhancement effect for different morphological ZnO nanostructures grown in microfluidic channels. Reprinted with permission from Sang et al., Biosens. Bioelectron. 75, 285–292 (2016). Copyright 2016 Elsevier. (c) A hierarchical 3D nanostructured microfluidic device for enrichment and sensitive detection of pathogenic bacteria. Reprinted with permission from Jalali et al., Small 14, 1801893 (2018). Copyright 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (d) Hydrogel microarrays entrapped acetylcholinesterase (AChE) and QD-Ag@Sillica for sensitive detection. Reprinted with permission from Kim et al., Biofabrication 10, 035002 (2018). Copyright 2018 IOP Publishing Ltd. (e) One-step immunoassays for enhanced detection of cardiac troponin I (cTnI). Reprinted with permission from Wang et al., Microsys. Nanoeng. 7, 65 (2021). Copyright 2021 the Author(s).

FIG. 2.

(a) Portable devices for the detection of multiple pathogens. Reprinted with permission from Yu et al., Small 13, 1700425 (2017). Copyright 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) The investigation of the fluorescence enhancement effect for different morphological ZnO nanostructures grown in microfluidic channels. Reprinted with permission from Sang et al., Biosens. Bioelectron. 75, 285–292 (2016). Copyright 2016 Elsevier. (c) A hierarchical 3D nanostructured microfluidic device for enrichment and sensitive detection of pathogenic bacteria. Reprinted with permission from Jalali et al., Small 14, 1801893 (2018). Copyright 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (d) Hydrogel microarrays entrapped acetylcholinesterase (AChE) and QD-Ag@Sillica for sensitive detection. Reprinted with permission from Kim et al., Biofabrication 10, 035002 (2018). Copyright 2018 IOP Publishing Ltd. (e) One-step immunoassays for enhanced detection of cardiac troponin I (cTnI). Reprinted with permission from Wang et al., Microsys. Nanoeng. 7, 65 (2021). Copyright 2021 the Author(s).

Close modal

In addition to the improvement in the detection efficiency for multiple targets, another promising direction of these plasmonic devices is to investigate and explore the lowest detection limit of such assays. Efforts have been devoted to developing optimal nanostructures with various morphologies and spacings to trigger large enhancement factors for high sensitivity.54 Sang et al. grew different morphological ZnO nanostructures in microfluidic channels and found that the sharp nanorod has the largest dynamic range and the strongest fluorescence intensity,68 as shown in Fig. 2(b). Although the MEF-based detection method achieved significant fluorescence enhancement through structural innovation, non-specific binding of proteins and the associated background noise render a low SNR and unsatisfactory detection sensitivity. A promising solution is to pre-concentrate target analytes in samples to increase the occurrence of binding events for the detection of analytes at lower concentrations. For instance, Jalali et al. proposed a microfluidics assay platform integrating hierarchical 3D nano/microisland (NMI) structures with nanorough protrusions for improved capturing efficiency and enhanced detection limit [Fig. 2(c)].56 In addition to passive enrichment technology, force fields, such as acoustics,57 electrics,69 or magnetics,70 generated by external sources have been also used to enrich analytes based on the biophysical properties of the biomarkers. More recently, the combination of both physical and biochemical methods has further improved the biomarker enrichment effect and shown potentials in real applications. In addition to various enrichment techniques, hydrogels and droplets have also been proposed to reduce the LoD in these microfluidic assay platforms integrated with plasmonic nanostructures. The detection of glucose and organophosphorus compounds has been achieved using hydrogel arrays containing metal nanoparticles, which amplified the fluorescent signal through MEF and yielded a significant enhancement of detection [Fig. 2(d)].58,71 As such, droplets serve as ultra-small volume reaction chambers for biological detection and chemical analysis. When combined with MEF, ultra-high-sensitivity molecular detection can be achieved.59,72 Another advantage of droplet-based assays is the ability to characterize target analytes at the single-molecule level, which enables the absolute quantification of biomolecules without external calibration. The combination of several techniques has been proposed to further ameliorate the sensitivity. For example, Xu et al. constructed a dual amplification fluorescence sensor by incorporating immunohybridization chain reaction (immuno-HCR) and MEF of carbon nanodots (CDs) for the detection of alpha fetal protein (AFP).60 We expect the limitations of sensitivity can be surmounted for quantifying the attomolar and zeptomolar antigen level with enrich-amplifying strategies.

The convenience and speed of immunoassays play an important role in point-of-care testing. Cumbersome and tedious operation procedures can be integrated into a single chip through the incorporation of microfluidics. The so-called one-step immunoassays have been developed, which include multiple sample preparation steps, such as liquid handling, mixing, and incubation for rapid and automated immunoassays. Interesting attempts have been made toward effective mixing, binding kinetics, and fluidic control over the samples. Among them, microwaves or surface acoustic waves have been demonstrated to accelerate antibody-antigen binding and shorten the incubation time.73,74 The concept of a one-step immunoassay chip has been implemented to integrate individual operation steps into a compact microfluidic chip.62,75 For instance, by merging a capillary pump, a deposition area, and a detection area on a microfluidic chip, Wang et al. have developed a one-step immunoassay for cardiac troponin I (cTnI) as presented in Fig 2(e).64 The automated, easy-to-use, and self-contained chip offers an opportunity for point-of-care testing, particularly in resource-limited regions.

The development of sensitive fluorescent immunoassay devices integrated with plasmonic nanostructures has revolutionized the current diagnostic technology. The progress in core technologies gave birth to niche applications for various detection purposes, as reviewed in the aforementioned parts. However, restrictions still exist to bring these devices into real applications. Here, we want to share our perspectives on several challenges to be addressed, as summarized in Fig. 3.

FIG. 3.

Challenges and future directions of MEF-based microfluidic assay devices.

FIG. 3.

Challenges and future directions of MEF-based microfluidic assay devices.

Close modal

First, significant challenges still need to be resolved to achieve stable and uniform metal-enhanced fluorescence over a large area. The effect of LSPR is greatly influenced by the thickness of the dielectric layer and nanostructure morphology. Precise models and theories are still required to predict the optimal structures for fluorescence enhancement. Another potential path to move forward is to develop new manufacturing techniques to fabricate nanostructures with high-throughput, excellent uniformity, high resolution, and low cost. Chemical synthesis is not compatible with the microfabrication process and faces issues of unsatisfactory uniformity. Lithography methods, although they allow a precise and uniform patterning of nanostructures, require expensive equipment and suffer from low throughput. Thus, it is of great priority that new fabrication techniques are introduced so as to easily integrate these nanostructures into assay chips for giant enhancement and robust detection results. The appropriate material selection of these nanostructures is also important for saving production costs. The most commonly used materials, namely, gold, silver, or other noble metals, exhibit significant fluorescence enhancement, yet at high costs. There remains a need for moderately priced materials with strong fluorescence enhancement effects. Zn-based55,68,76,77 and Al-based11,78 materials have been demonstrated recently for fluorescence detection owing to their low costs and easy fabrication methods, which pave the way for further investigations on nanofabrication.

Regarding the microfluidic assay technology, human biological specimens, including blood, saliva, and urine, are intricate and comprise a plethora of biomarkers and pathogens associated with different diseases and biological properties. Hence, the simultaneous detection of multiple biomarkers in complex specimens on a single microfluidic chip is paramount for the early detection of diseases with high efficiency.79 The multi-analyte detection techniques highlighted previously are achieved by the arrangement of microfluidic channels in a parallel configuration, yet facing the challenge of cross-reactivity or fluorescence interference within a shared chip substrate. As a consequence, meticulous and deliberate design considerations become imperative to secure the precise discrimination of distinct analytes. In addition, owing to the intricate nature of biological samples, appropriate pre-treatment steps are required before analysis, such as hydrolysis of hair and or nucleic acid amplification to extract target analytes. High sensitivity and specificity can be also achieved through sample pre-concentration and reduction of non-specific binding incorporated with hydrodynamic, acoustic, electrical, magnetic, and other technologies. On the detection side, conventional optical modules for fluorescent analysis are relatively bulky and expensive. The utilization of smartphones integrated with microfluidic assay chips has been proposed as an alternative for these bulky fluorescent immunoassay analyzers with advanced imaging setups and enormous computational power.80–82 Moreover, machine learning or neural networks have become popular for forecasting the testing results from intertwined sensing information. High-throughput microfluidic assay platforms provide abundant data to train artificial intelligence models for precise and automated diagnostics. Another issue may be the limitation of fluorescent detection, which requires complicated labeling techniques and is confronted with photobleaching or damage to cells. In this case, label-free detection techniques may replace the existing fluorescent strategies for next-generation bioassays.83 

In summary, we have reviewed recent studies that integrate plasmonic nanostructures into microfluidic assay devices for sensitive detection of biomarkers. Tremendous efforts have been made toward addressing practical issues encountered in fabrication, stability, efficiency, automation, and accuracy. The potential opportunities and future trends for new technology development are also discussed. We envision that technological breakthroughs can be accomplished to tackle the existing challenges and finally to implement a truly integrated system for rapid and accurate diagnosis of diseases.

This work was financially supported by the National Key R&D Program of China (No. 2021YFB3602200), the National Natural Science Foundation of China (NNSFC) (No. 62171403), the Natural Science Foundation of Zhejiang Province, China (No. LZ22F010002), and the Fundamental Research Funds for the Central Universities (No. 226-2022-00154).

The authors have no conflicts to disclose.

Xuefeng Xu: Data curation (equal); Formal analysis (equal); Writing – original draft (equal); Writing – review & editing (equal). Guangyang Li: Writing – review & editing (supporting). Lingyue Xue: Writing – review & editing (supporting). Shurong Dong: Resources (equal); Supervision (equal); Writing – review & editing (equal). Jikui Luo: Resources (equal); Supervision (equal); Writing – review & editing (equal). Zhen Cao: Conceptualization (equal); Funding acquisition (equal); Investigation (equal); Resources (equal); Writing – original draft (equal); Writing – review & editing (equal).

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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