EMERGING PLATFORMS FOR HIGH-THROUGHPUT ENZYMATIC BIOASSAYS

Abstract

Enzymes play essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in-vitro enzymatic bioassays have been developed for use in industrial and research fields such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many requires the aid of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity remain to be resolved. Herein we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights on remaining challenges and future research trends.

Significance of Enzymatic Bioassays and Development of High-Throughput Platforms

Enzymes are biocatalysts that accelerate biochemical reactions in living organisms and are critical for modulating many important systems required for life. Since the 1960s, scientists have been developing high-throughput enzymatic bioassays for a wide range of important applications in both industrial and research fields, including enzyme activity in cell biology, enzyme engineering, drug discovery in pharmaceutical industry, biofuel production and vaccine adjuvant development [1–6]. For example, drug discovery processes often focus on screening candidate molecules targeting key enzymes because many diseases are associated with dysfunctions in enzyme catalysis [7, 8]. Moreover, understanding the bioactivity, stability, specificity, kinetics, and tolerance of enzyme variants can not only help study the biological mechanisms but also facilitate the engineering of desired functionalities for applications on demand [9–11]. The various applications of high-throughput enzymatic assays can be roughly categorized as analytical analysis of analytes, enzyme or enzyme secreting cells screening, enzymatic kinetics characterization, and enzyme inhibitor and drug screening (Box 1). Regardless of the category, these enzymatic bioassays typically involve the aid of high-throughput platforms including high-throughput screening that typically involve a large library of candidate compounds or digitization that digitizes the reaction into millions of reaction chambers or droplets in high-throughput. Therefore, it is critical to advance high-throughput platforms to further drive this field forward. In this review, we aim to present an up-to-date overview of notable high-throughput platforms for performing enzymatic bioassays. Among them, microtiter plate is the mature standard platform that remains popular but faces some critical challenges in reagent consumptions and limited scalability. In recent years, microfluidic array and droplet microfluidics have emerged as promising high-throughput platforms to address the shortcoming of microtiter plate (Figure 1, key figure). Here we summarize the analytical performance of various microfluidic platforms using a comprehensive list of criteria, including the reagent consumption to evaluate the cost, reaction manipulation feasibility for multi-step bioassays, reactant recovery for further downstream analysis, real-time analysis for kinetics measurement, concentration gradient range and multiplexing feasibility for overall throughput and scalability. Of note, we only discuss microfluidic platforms that perform homogeneous reactions without consideration of diffusion-limited models [12]. Moreover, we outlined the remaining challenges and opportunities that are important to facilitate the growth of this nascent field as well as the trend. We hope this review can serve as an overview for high-throughput platforms toward enzymatic bioassays and provide the readers a guidance for designing the appropriate tool in practice.

Box 1. Applications of high-throughput enzymatic bioassays.

Analytical analysis of analytes Compared with traditional diagnostics and analytical methods, enzymatic bioassays showed great potential for qualitative and quantitative analysis of different analytes in biomedicine and clinical diagnosis. These include such as matrix metalloproteinase (MMP)-9 detection of breast cancer cells for studying extracellular vesicle exosomes and tumor metastasis via commercial assay kit [16], urea measurement screening via immobilized alginate-chitosan enzyme biosensors [17], and viable bacteria counting based on the fluorescence change of resazurin when reduced by coenzyme NADH secreted from viable bacteria cells [28], due to its high sensitivity, specificity, and potential for mass production.

Enzyme or enzyme secreting cells screening: Using enzymatic bioassays to screen and isolate out novel enzymes or enzyme-producing cells is another important application for the pharmaceutical and biofuel industry, where researchers have done transaminase screening [97], lipase-producing bacteria screening [34], directed evolution [23, 57], secretory phenotype screening [71], and esterase isoenzyme screening [56].

Enzymatic kinetics characterization: To better understand and develop more efficient enzymes, enzymatic activity assays as another type [43, 47, 65, 76, 77, 82, 87] have been employed to characterize the enzyme kinetics and its biological functions in a high-throughput manner.

Enzyme inhibitor and drug screening: Enzymes have also been widely used as the target or labeling for screening the potential inhibitors or drugs [15, 37, 88], and vaccine candidates [55], due to its essential catalytic activity and its vulnerability to be modulated by drugs [7].

Key figure: Figure 1. Emerging platforms for high-throughput enzymatic bioassays.

Versatile technologies using microfluidic array and droplet microfluidics have been developed to aid the high-throughput enzymatic bioassays. Based on the functionality of platforms, microfluidic array is further classified as microwell array and contact printed array, and droplet microfluidics is classified as binary detection with integrated sorting, kinetic seeing via continuous flow, real-time measurement via in-situ trapping, and multiplexing and combinatorial screening. Each platform has their own characteristics among the others, and this review summarize the analytical performance of each and provide the future perspectives to advance this field.

1. Microtiter Plate

We begin with microtiter plate – the standard high-throughput platform – as the reference for comparing with emerging high-throughput platforms. To date, microtiter plates and plate readers are still often employed for performing high-throughput enzymatic assays in academic research (Table 1), such as the discovery of active and enantioselective amino alcohol-specific transaminases [13], discovery of biological H₂ producing hydrogenase and ferredoxin-NADP⁺ reductases [14], and screening for apyrase activity [15]. Some research efforts have focused on developing compatible accessories for enhancing the performance of microtiter plate assays. Examples include a PDMS (see Glossary) concentrator that can be inserted into a microwell to electrokinetically concentrate enzymes, substrates, and fluorescent reaction products, and thereby increase the assay reaction rate [16], and a lid with immobilized enzymes that allows immediate initiation and stoppage of the enzymatic reaction by simply flipping the microtiter plate [17]. But the major advance of microtiter plate lies in integration with automated robotic systems and high-speed computers. For example, Dörr and colleagues demonstrated a fully automated platform consists of a central robotic arm to transport microtiter plates from site to site, a pipette robot to dispense liquid samples in a rapid and precise manner, a plate reader, incubation shakers and a carousel for storage, which enabled high-throughput enzymatic bioassay precisely without manual procedures [18]. Another example is Molecular Screening Shared Resources – an open-access high-throughput screening (HTS) laboratory in UCLA that use robotics-integrated microtiter plate platform to reach a screening capacity of > 100,000 compounds a day [19]. These examples illustrate advantages of microtiter plate in manipulating, titering, multiplexing, and recovering reactions (enabled by robotics) and performing real-time measurements (enabled by plate readers). However, it is still a challenging task to perform large-scale high-throughput enzymatic bioassays using microtiter plates due to the high reagent consumptions in microliter level and exponentially increased assay number with the candidates in the library, which prevent these platforms from screening compounds in million level [20].

Table 1.

Summary of recent advances of using microtiter plate for high-throughput enzymatic bioassays

Microtiter Plate	Key Concept or Principle	Application/Demonstration	Throughput: Well Number	Ref
	Tetrazolium red (TTC) based colorimetric enzymatic assay	Transaminase activity and enantioselectivity detection	96-well	[13]
	High-throughput platform for measuring biological H2	H2 production screening to find CPI hydrogenase and FNR mutants	96-well	[14]
	Low-volume luminescence-based enzymatic assay	Apyrase activity detection	384-well	[15]
	PDMS concentrator and microtiter integration	Screening matrix metalloproteinase (MMP)-9 expression	12-well	[16]
	Robolids microplate for immobilized enzyme-based assays	Measurement of urea in human urine and serum	96-well	[17]
	Fully automatized high-throughput enzyme library screening platform	Screen and identify novel enzyme variants	96-well	[18]

2. Microfluidic Array

Compared with the conventional microtiter plate based-high-throughput technologies, the microfluidic array technique can reduce reagent consumption by orders of magnitudes [21, 22]. In particular, the reagent consumption per assay lies in the pico/nanoliter level and the throughput can reach up to tens of thousands on a chip (Table 2). Based on the generation method and principle, we categorized the microfluidic array technique into microwell array and contact printing array, where the former one is realized by loading the reactants into confined microwells and the latter one is implemented by dispersing the reactants on a semi-open substrate.

Table 2.

Summary of recent advances of using microfluidic array formats for high-throughput enzymatic bioassays

	Key Concept or Principle	Application/Demonstration	Reaction Volume	Throughput: Number of reactions per cm2	Conditions per Experiment	Ref
Microwell Array	Femtoliter droplet array (FemDA)	Enzyme mutant acquisition, digital enzyme assay, single-molecule analysis	fL	1,000,000	1	[23] [24] [25] [26]
	Multilayer microfluidic array	DNA methylation and enzymatic assay	750 pL	13,000	1	[27]
	Picoarray	Viable bacteria counting	250 pL	1,200 *	5	[28]
	Microwell array with single enzyme activity-based protein profiling (SEAP)	Enzyme characterization	N.D.	1,750,000 *	1	[29]
	Equant screw valve-based (SIES) microfluidic chip	Digital PCR and enzymatic assay	1.25 nL	140 *	1	[30]
	Microfluidic Chip for multi-step assay	Nonhormonal contraceptive agent screening	N.D.	1 (Scalable) *	3	[31]
	Agarose-based microwell array	Screening functional microorganisms via Microbial enzymatic activity assay	3 pL	150,000	1	[34]
	Key Concept or Principle	Application/Demonstration	Reaction Volume	Throughput: Number of droplets per array	Conditions per Experiment	Ref
Contact Printed Array	Micropipette with unilateral Taylor-Aris dispersion-based dilution	Quantitative HTS (qHTS)	8 nL	625 *	2,448	[37]
	Contacted printed array with Desorption electrospray ionization mass spectrometry (DESI-MS)	Acetylcholinesterase assay, N-alkylation and Suzuki coupling reactions, nucleophilic aromatic substitution reactions.	50 nL	64 *	384	[40] [41] [42]
	Dipping-depositing-moving (DDM)	Single-cell enzymatic activity assay	179 pL – 1.8 nL	1,700 *	4 to 7	[43]
	Double inkjet printing	Single cell culture and single molecular enzymatic assay	100 – 500 pL	450 *	6	[45]
	Microfluidic streak plate	High-throughput microbial cell separation and cultivation	180 pL – 6 nL	24 *	3	[46]
	Nanoliter-printed droplet array	HTS of cell-secreted proteins	7 nL	485 *	5	[47]

N.D. refers to Not Defined.

^*Indicates the estimated value based on the literature.

2.1. Microwell Array

Microwell arrays digitize the reaction contents into tens of thousands of femto- to nanoliter microwells to perform high-throughput and parallelized reactions. The low reagent consumption for each assay and real-time analysis can be achieved, though it has relatively lower feasibility for reaction manipulation, recovery, multiplexing, and lower concentration range. As a representative example, recently Zhang and colleagues proposed a massively parallel femtoliter droplet array (FemDA) using the trapping concept to conduct cell-free protein synthesis (CFPS) [23]. Each FemDA was designed to contain 10⁶ uniform femtoliter microwells per cm², where each droplet or reaction was trapped in the microwells formed with a hydrophilic bottom (glass) and hydrophobic barrier (CYTOP, a perfluoropolymer) and digitalized with a two-step oil sealing strategy (Figure 2A). Their group has also demonstrated digital enzyme assay [24] and single-molecule analysis [25, 26] based on a similar microwell array. Though FemDA achieved small femtoliter reaction volume and high array density, it can only test one condition at a fixed concentration and lacks the means for retrieving a positive reaction.

Figure 2. Representative platforms for (A) Microwell array, (B) Contact printed array, (C) Binary detection with integrated sorting, (D) Real-time measurement via in-situ droplet trapping, (E) Multiplexing and combinatorial screening.

(A) A massively parallel femtoliter droplet array (FemDA) traps reaction into the microwells formed with a hydrophilic bottom (glass) and hydrophobic barrier (CYTOP, a perfluoropolymer), with two oil sealing strategy [23]. Image waiting for permission from [23]. (B) A nanoliter quantitative high-throughput system with a unilateral Taylor-Aris disperser, where droplets can be printed into an well with the aspiration of the sample and diluent [37]. Image adapted with permission from [37]. (C) Workflow of a high-throughput droplet microfluidics platform combining a nanoelectrospray ionization-mass spectrometry (nESI-MS) and DEP sorting. In their system, each mother droplet was split into two daughter droplets, one flowing into a PFA capillary measured by ESI-MS and the other one travel into a delay line to allow time to get the sorting instructions from the ESI-MS [66]. Image waiting for permission from [66]. (D) Schematic and workflow of a droplet-based microfluidics platforms with pico-injector that can realize kinetic measurement via six measurement points [76]. Image waiting for permission from [76]. (E) Schematic and workflow of a novel microcage concept-based droplet trapping system, where the microcage design ensures smooth oil spreading during the droplet suspension spreading process [80]. Image adapted with permission from [80]. (F) Schematic and workflow of the a barcode-free combinatorial screening platform that uses pneumatic-valve to programmatically control the injection of different substrates and MMPs with in-line incubation and detection [86]. Image adapted with permission from [86].

Improvements on the typical trapping concept-based microwell array focused on enhancing the loading efficiency to reduce the reagent waste and enable the testing of multiple conditions and multiple enzymes. O’Keefe and colleagues have demonstrated a multilayer microfluidic array that achieved highly efficient sample loading into 13,000 picoliter microwells [27]. The curved channel design and optimized well-height to channel-height ratio helped direct the fluids towards the microwell inlets to enhance the loading efficiency. Hsieh and colleagues presented a Resazurin-Amplified Picoarray Detection (RAPiD) that allowed the digitization of reactants into 5 channels of 1,400 picoliter microwells for on-chip bacteria culture and detection [28]. Sakamoto and colleagues have demonstrated single enzyme activity-based protein profiling (SEAP) that can characterize multiple enzyme species using microwell array based on different colored and structure-wide substrates [29].

Typical microwell arrays show great performance for homogeneous assays but were generally incapable of performing multi-step assays that require multiple sample loading steps. Hu and colleagues overcame this limitation with their proposed self-priming isometric and equant screw valve-based (SIES) microfluidic chip, which implemented step-by-step sample loading by designing a water tank to balance the negative pressure of the chip and a screw valve to control the vacuum-based sample loading steps [30]. Li and colleagues developed a scalable multiwell microfluidic device to perform a multi-step assay that requires cell incubation, enzymatic removal of cumulus cells, washing, and staining, demonstrated by screening nonhormonal contraceptive agents [31].

The majority of materials used for microwell arrays such as poly(ethylene glycol) diacrylate (PEGDA) microwells [32] and silicon microwells [33] were not feasible to continuously provide nutrients to screen living targets of interest. In response, Zhang and colleagues proposed a fluorescent dye mixed-agarose-based microwell array chip to screen microorganisms by permitting continuous diffusion of nutrients into the picoliter microwells [34]. Such design enabled the real-time monitoring of cell growth and cell metabolism.

2.2. Contact Printed Array

Contact printing is commonly used to create high-throughput assays by printing desired reactants at the nanoliter level in a controlled manner on a semi-open platform [35, 36]. The recovery, manipulation, real-time analysis, high concentration gradient range, and multiplexity of the reaction can be achieved, though it has relatively large reagent volume in nanoliter and limited array density. As a representative example, Wei and colleagues have developed a nanoliter quantitative high-throughput system to study enzyme kinetics and screen enzyme inhibitors, which was performed by printing 2,448 12-nL reactions through a unilateral Taylor-Aris disperser (Figure 2B) [37]. Their system showed the capability to achieve a tunable range of concentration gradients covering 6 orders of magnitude versus the 2 to 4 orders of magnitude covered by the conventional methods of manipulating the merging flow rate or merging the droplets with dilution droplets in a microwell [38, 39].

Mass spectrometry was also demonstrated to be feasible for integration with contact printed array to enable direct analysis from complex enzymatic bioassay metrics. For example, Cooks’ group adopted desorption electrospray ionization mass spectrometry (DESI-MS) for analysis and monitoring of thousands of 50-nL printed reactions on a glass slide, demonstrated with acetylcholinesterase assay [40], N-alkylation and Suzuki coupling reactions [41], and nucleophilic aromatic substitution reactions [42].

Such micropipette or dispersion-based approach, however, in general is limited by the low droplet density because of the relatively large droplet volumes in nanoliter and the complexity of the operation procedures. In response, Guo and colleagues proposed an automated system that applied the dipping-depositing-moving (DDM) concept to generate femtoliter to picoliter droplets on an oil-covered hydrophilic micropillar array chip, demonstrated with kinetic matrix metalloproteinases (MMP-9) analysis and single cell enzymatic activities [43].

Inkjet printing has become another promising approach to achieve the direct generation of picoliter or sub-nanoliter reactions [44, 45]. The recent development of inkjet printing techniques lied in addressing the evaporation issue, which limits its wide use toward enzymatic research. Sun and colleagues have developed a picoliter reaction array for single molecular enzymatic assays based on a novel double-inkjet printing method that used piezoelectric inkjet printing equipment to first generate oil droplets of defined volumes on the planar substrate followed by precise delivery of the reagents into the oil droplets by jet action [45].

Other than printing the reactants directly on the substrate, many groups have also integrated the pre-generated droplets with contact printing methods to generate an open-surface platform that overcame the challenges of droplet manipulation and recovery using the conventional enclosed droplet array. Jiang and colleagues created a high-throughput single-cell cultivation on microfluidic streak plates [46]. In their platform, droplets were transported via tubing and dispensed onto salinaied surface to form the array. Haidas and colleagues also coupled a similar dispensing system to generate droplets on the indium tin oxide (ITO)-coated hydrophilic plates, which showed the feasibility for additional manipulation of the droplet content [47].

3. Droplet microfluidics

Droplet-based microfluidics in recent years showed great progress toward high-throughput enzymatic bioassays [48–52]. Compared with the microfluidics system with continuous fluid flow, droplet-based microfluidics utilizes the water-in-oil emulsion droplets to compartmentalize reactions into massive nano- to femtoliter droplet reactors, which fulfill the needs of digitizing enzymatic reactions with high-throughput [53, 54]. Moreover, such small volume reactors and parallelized experiments can enhance the signal-to-noise ratio, shorten the assay time, and reduce the reagent consumptions. These advances in droplet microfluidics have enabled it toward versatile applications and functionalities, which can be classified as the sorting of large number of reactions with binary detection, kinetics screening, real-time measurement, multiplexing and combinatorial screening.

3.1. Binary detection with integrated sorting

One of the biggest accomplishments of droplet microfluidics is the ability to screen a large amount of pre-digitized picoliter droplets using passive droplet generation method and select specific targets of interest with binary detection [48], which is essential in applications such as sorting HIV particles, esterase isoenzymes, and directed enzyme evolution [55–57]. Such approach showed high screening throughput, low reaction volume, and reactant recovery capability, though in general droplet sorting has low reaction manipulation and multiplexing capabilities due to the identical droplet content. The optical signal from the droplets is a commonly used fingerprint for screening, which determines the subsequent actuation on the droplets. There are various mechanisms to actuate droplets with defined parameters and thereby realize droplet selection and recovery, such as electric [58], acoustic [59], magnetic [60], pneumatic [61], and thermal-based [62] actuation mechanisms. Electric actuation among them represents a robust mechanism with high sorting frequency (e.g., up to 30 kHz reported recently [63]), accuracy, and selectivity.

Other than different sorting mechanisms, various detection systems have been integrated with droplet sorting. Among them, mass spectrometry (MS) was proved to be a good addition to droplet sorting because MS offers universal label-free detection with high sensitivity and selectivity, as well as the flexibility for multiplexing [64]. As a representative example, Steyer and colleagues have demonstrated the feasibility of coupling a high-throughput droplet microfluidics platform with a nanoelectrospray ionization-mass spectrometry (nESI-MS) for stable long-term analysis and operation [65], which were further coupled with dielectrophoresis (DEP) sorting to enable high throughput enzymatic screening and sorting [66] (Figure 2C).

Alternatively, fluorescence-activated droplet sorting (FADS) is one of the most used sorting methods that utilizes electric fields to alter the motion of the “positive” droplets with a specific fluorescence signal threshold that is encapsulated with the enzyme and the corresponding cells (or other components for cell-free systems) [67–69]. Recently Chaipan and colleagues presented an ultrahigh-throughput (500 Hz) droplet-based microfluidic platform that screened and sorted millions of single virus particles with high sensitivity to find the vaccine candidates with the best antigenic features [55], which was still challenging with such small size of particles or low amount of surface proteins.

As most target molecules in industrial HTS are small-molecule pharmaceuticals that are not feasible to adopt into fluorescent enzymatic assays, tremendous efforts have been made to apply FADS for screening of diverse types of enzymatic reactions and assays, [56, 66]. Absorbance-activated droplet sorting (AADS) technique has demonstrated the capability to extend the range of the assays [57]. Gielen and colleagues developed an AADS-based system for sorting of directed enzyme evolutions with ultrahigh throughput (100 Hz) that broadened the scope of the directed evolution assays from fluorescent reaction products to those producing UV/Vis active chromophores [57].

In addition to the progress in the detection systems to couple with droplets sorting, modifications to the assay design and sorting criteria have been proposed to further broaden the range of the assays. Abatemarco and colleagues proposed an RNA-aptamers-in-droplets (RAPID) technique to perform FADS that used “spinach (an RNA sequence that binds and activates the fluorescence of a small-molecule fluorophore [70])” aptamers to convert the non-fluorescent extracellular products into fluorescence signals [71]. Horvath and colleagues achieved label-free enzyme sorting by interfacial tension (SIFT) [56], where the droplets with different pH, such as esterase, lipases, and dehalogenases, can result in different interfacial tensions [72].

3.2. Kinetic screening under continuous flow

Enzyme kinetics and inhibition are essential for studying a wide range of disciplines such as pharmacology, medicine, and industrial biofuels production [73, 74]. The conventional methods such as microtiter plates and microfluidics arrays can be difficult to study fast enzymatic reactions because of the low time resolution in the minute scale [75]. Therefore, droplet microfluidic platforms have been developed to achieve the kinetic screening of a large amount of pre-digitized picoliter droplets in continuous flow and a timely manner, with innovations on the detection system. Such approach in general showed the low reaction volume, high screening throughput, and kinetic measurement capability, while facing challenges on the multi-step assays, content manipulation, and reactant recovery. As a representative example, Sjostrom and colleagues have presented a droplet-based microfluidic platform integrated with a droplet picoinjector that can realize kinetic measurement for all assay conditions [76]. Replying on the six designated measurement points downstream, the measurements were later used to extract the reaction velocities for the mixture of substrate and different inhibitor concentrations, which in turn determined the kinetic parameters of the enzyme substrate inhibitor system (Figure 2D). However, despite the kinetic screening capability and high throughput of 840 Hz of the injection, their platform can only test one condition at a fixed concentration and lack of means for retrieving a positive reaction.

Many groups have also tried to integrate droplet microfluidics with wide field fluorescence imaging systems to detect a homogeneous set of droplets [75]. However, the wide field fluorescence detectors limit the detection of droplets moving at high linear velocities especially when utilizing long exposures, making it difficult to monitor a reaction along an extended microchannel. Hess and colleagues combined the droplet-based microfluidics and a wide field stroboscopic illumination to introduce microsecond excitation pulses to increase the temporal and spatial resolutions and reduce the motion blur from the reduced exposure time (reduced from millisecond scale down to 20 μs) and high droplet velocities (>5 cm/s) [77].

3.3. Real-time measurement via in-situ droplet trapping

Real-time kinetic screening with high throughput is still a challenge for droplet microfluidics with continued channels. Among different platforms, microfluidic array and microtiter plate showed great feasibility to enable real-time analysis by physically immobilizing the reactions [45, 47, 78, 79]. Inspired by the similar concept, in-situ droplet trapping has been proposed to immobilize the pre-generated droplets onto the array, where indexing and monitoring of the droplets over time can be achieved [78]. Such approach enabled the real-time analysis feasibility that general droplet microfluidic systems cannot achieve, despite the challenges on the multi-step assay and content manipulation with the pre-generated droplets, and the droplet recovery. As a representative platform, Xu and colleagues have developed and adopted a novel microcage concept to trap the droplets with 100% droplet recovery. Those microcages were surrounded by micropillars and the gaps between the micropillars can serve as distribution channels to spread the oil from the cage during the droplet suspension spreading process. Such design can avoid the resistance of droplet trappings in the conventional microwell design, where the opposite flow direction of the droplets and the oil, where the droplets move into the well and the oil moves out from the well (Figure 2E) [80]. Their semi-open system achieved the reactant recovery that most closed systems cannot achieve and enabled real-time analysis, though with limited reaction manipulation capabilities to the preformed droplets.

Many alternatives in-situ trapping designs have been proposed to overcome the increased resistance with the commonly used entrapment structure-based flow trapping design when flowing high density of droplets [78, 79]. Labanieh and colleagues have developed a dual-layered floating droplet array (FDA) device using the concept of droplet buoyancy [78]. The floating droplets were trapped into the microwells on the PDMS ceiling, which were recovered by simply flipping the chip to refloat the droplets back to the flow channel. Um and colleagues developed another droplet trapping device that incorporated a mesh-grid to achieve the easy trapping and merging of the droplets, which were used to study cell-to-cell communications [81]. O’Keefe and colleagues proposed a real-time droplet analysis platform that adopted a novel “pseudosieve floor” with tightly spaced posts to spread the oil and enhance the droplet sedimentation and a “highway design” without posts to release the back pressure [79].

Mass spectrometry was also demonstrated to be feasible for integration with in-situ trapping. Heinemann and colleagues integrated a droplet array with a nanostructure-initiator mass spectrometry (NIMS) in a so-called platform - μNIMS, which enabled the digital control of cellulose-degrading enzymes activity with electrowetting-on-dielectric (EWOD) and the sensing and detection with NIMS [82].

3.4. Multiplexing and combinatorial screening

Another key aspect of high-throughput enzymatic bioassays is to achieve multiplexing and combinatorial screening. Advances on the droplet microfluidics such as active or valve-based generation method and assay modification have enabled the multiplexing and combinatorial screening capability. Those approaches in general showed high content manipulation and multiplexing feasibility, while require a relatively large reagent consumption in nL scale and low throughput. As representative examples, Wang’s lab adopted pneumatic valves to generate droplets with different composition combinations in an automated manner (Figure 2F) [83, 84] and further integrated with flowing focusing junction to achieve picodroplet content customizability, which was named the Integrated Programmable Picodroplet Assembler (iPPA) [85, 86]. By controlling the opening and closing stage and time of multiple sample loading inlets, the nanoliter droplet contents can be easily manipulated with orders of magnitude combinations, which can be in-line discretized into picoliter droplets. The monodisperse picoliter droplets thus also gain reaction manipulation and multiplexing capacity for combinational screening. However, although iPPA solves the reaction manipulation and multiplexing limitations of most passive and flow focusing based-droplet microfluidics systems, it lacks the capability of droplet recovery and real-time analysis.

Other than the improvements on the screening systems, modifications on the assay achieved multiplexing ability to analyze multiple analytes simultaneously, proposed by Ng and colleagues [87]. More specifically, they modified the FRET (fluorescence resonance energy transfer)-substrate to accommodate different sets of fluorescence pairs with respective excitation and emission wavelengths so that multiple biological aspects of the enzyme can be studied simultaneously in one single droplet.

Creating a large library of droplets containing combinatorial compounds is still challenging for drug screening. In response, Ochoa and colleagues integrated high-performance liquid chromatography (HPLC) with droplet-based microfluidics to perform bioactivity-guided assays in droplets containing HPLC-separated chemical compounds [88]. Moreover, high-speed electrocoalescence or picoinjector techniques have been employed to generate droplets with multiple reagents for combinatorial screening [89, 90].

Concluding Remarks and Future Perspectives

High throughput platforms play important roles in enzyme engineering in industrial and research needs. In this review, we summarized the platforms in three categories according to the unique advantages in different applications, including the microtiter well plate, microfluidic array, and droplet microfluidics. We analyzed the performance of these platforms from multiple perspectives, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity (Table 4).

Table 4.

Summary of different high-throughput platforms regarding their analytical performance and research directions.

		Microfluidic Array		Droplet Microfluidics
	Microtiter Plate	Microwells	Contact Printing	Binary Output	Kinetic Screening	In-Situ Trapping	Multiplexing
Reagent Consumption	μL	fL - nL	pL - nL	pL	pL	pL - nL	nL
Manipulation Feasibility	High	Low	High	Low	Low	Low	High
Single Reactant Recovery	Yes	No	Yes	Yes	No	No	No
Real-time Analysis	Yes	Yes	Yes	No	Partially	Yes	No
Concentration Range	High	Low	High	Low	Low	Low	Medium
Multiplexing Feasibility	High	Low	High	Low	Low	Low	Medium
Research Direction	■ Robotics ■ System Integration ■ Assay Modification	■ Multi-step Assay ■ Reactant Recovery	■ Reagent Volume ■ Array Density	■ Multi-step Assay ■ Content Manipulation	■ Multi-step Assay ■ Content Manipulation ■ Reactant Recovery		■ Throughput ■ Scalability

Red, orange, and green color respectively refers to increasingly better performance.

We foresee the continued use of microtiter well plates in the industry due to the mature pipelines with automated, multiplex, and stable procedures. Microfluidic array can reduce the reagent consumptions by orders of magnitude and show potentials for high-throughput enzymatic bioassays (10³ to 10⁶ reactions per array). Moreover, it in general facilitates high-throughput kinetic screening. Droplet microfluidics with continued microchannels as another high-throughput platform avoided the limitation from the small footprint of the device, which ideally can screen unlimited input droplets.

Despite the promising performance of those platforms, more investigations are needed to overcome their current limitations (see Outstanding Questions). We foresee future improvements on the integration with robotic systems, thermal controllers, or other accessory systems to improve its flexibility and assay performance such as sensitivity and reaction time. Microwell array as one array format showed higher reaction density on the array and lower reagent consumption, but the future direction should be focusing on the multi-step assay (e.g. SIES chip [30]) and reactant recovery. On the contrary, contacted printed array as a semi-open surface platform proved to be able to manipulate the reaction contents, generate a high concentration gradient range, and recover the reactants, but more investigations need to solve the restriction of using relatively large volume to increase the array density (e.g., FemDA [23]). Additionally, more questions or investigations for droplet microfluidics need to focus on the ways to conduct multi-step assay, manipulate droplet contents for heterogeneous assays, reactant recovery, throughput, and scalability for industrial uses. Lastly, the current droplet microfluidic systems current still rely on the use of tubing as the sample-to-chip interface [91, 92], such that each sample is injected through a designated tubing and inlet into the device via a pressure source such as a syringe pump, which hinders the scalability of droplet microfluidics to achieve the screening of a large compound library. Further research of novel sample-to-chip interfaces (e.g., autosamplers [93–95] and tubing-free interface [96]) can push droplet microfluidics a step closer to industrial-level screening.

Outstanding Question Box.

• How to integrate microtiter plates with accessory systems to enhance the performance of microtiter plate assays such as sensitivity and assay reaction rate.

• How to perform multi-step assays using a microwell array and retaining the target of interest?

• How to increase the assay throughput and avoid evaporation using the contact printing method?

• How to use droplet microfluidics to conduct multi-step assays? More specifically, how to load reagents or samples into the pre-formed pico/nanoliter droplets to initiate multi-step biochemical reactions at a given time/droplet position?

• How to manipulate individual pico/nanoliter droplets in a droplet cluster for the selection of specific targets of interest?

• How to achieve the combinatorial screening using droplet microfluidics in a high throughput manner?

• How to interface a large compound library with microfluidics device for industrial-level screening?

Table 3.

Summary of four types of droplet microfluidics systems for high-throughput enzymatic bioassays

	Key Concept or Principle	Application/Demonstration	Reagent Consumption	Throughput: Droplet Generation Frequency (Hz)	Conditions per Experiment	Ref
Binary Output	Nanoelectrospray ionization-mass-spectrometry	Cellular secretion monitoring and enzymatic variant evaluation	>65 pL	10	1	[65]
	Mass activated droplet sorting (MADS)	Enzymatic assay screening (not compatible with FADS- or AADS- based methods)	25 nL	0.7	1	[66]
	Fluorescence-activated droplet sorting (FADS)	Neutralizing epitopes screening on HIV particles for vaccine candidate	30 pL	500	1	[55]
	Absorbance-activated droplet sorting (AADS)	Directed enzyme evolution	180 pL	100	1	[57]
	RNA-aptamers-in-droplets (RAPID)	Secretory phenotype screening	pL	N.D.	1	[71]
	Enzyme sorting by interfacial tension (SIFT)	Esterase isoenzyme sorting	N.D.	30	1	[56]
Kinetic Screening	Droplet microfluidics with multiple measurement points	Enzyme kinetics and inhibition	pL	840	1	[76]
	Wide field stroboscopic illumination with droplet microfluidics	Enzyme reaction kinetics	pL	150	1	[77]
Multiplexing and Combinatorial Screening	Barcode-free combinatorial screening	Enzyme-substrate screening	nL	N.D.	650	[83]
	Integrated programmable picodroplet assembler (iPPA)	Single cell-based enzymatic screening, Single cell antibiotic susceptibility test	nL	N.D.	34	[85] [86]
	Droplet microfluidics with multi-color FRET substrate	Single cell multiplexed assay	pL	4,000	6	[87]
	Droplet-based microfluidics coupled with HPLC	Enzyme inhibiting assay	nL	8	3	[88]
	Key Concept or Principle	Application/Demonstration	Reagent Consumption	Throughput: Number of reactions per cm2	Conditions per Experiment	Ref
In-situ Droplet Trapping	Microcage-based large scale droplet array	Single cell enzyme activity assay	33 pL	33,000	1	[80]
	Dual-layered floating droplet array	Enzymatic screening assay	1.25 nL	16,000	1	[78]
	Mesh-integrated microdroplet array for single-cell droplet merging	Cell to cell communication	10 pL	22,500	1	[81]
	Real-time droplet analysis platform	Real-time enzyme kinetics, PCR, Digital melt curves	120 – 1000 pL	17,050	1	[79]
	Integrated microfluidics with NIMS (μNIMS)	Digital control of cellulose-degrading enzyme activity	8 nL	N.D.	1	[82]

N.D. refers to Not Defined.

Highlights.

• Advances in microtiter plates, microfluidics arrays, and droplet microfluidics showed unique advantages for high-throughput enzymatic bioassays regarding the key performance metrics including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity.

• Advances in microtiter plates focus on the integration with accessory systems, trying to improve the overall performance such as sensitivity and reaction rate.

• Advances in microfluidic array try to reduce reagent consumption, increase assay throughput, enable multi-step bioassays and reaction recovery.

• Advances in droplet microfluidics enables binary detection with integrated sorting, kinetic screening under continuous flow, real-time measurement via in-situ droplet trapping, and multiplexing and combinatorial screening.

Glossary

Absorbance-activated droplet sorting (AADS)

Droplets being sorted in a absorbance-activated manner

Aptamer

An oligonucleotide or peptide molecule that binds to a specific target molecule

Electrocoalescence

The coalescence of droplets under the influence of a variable electric field

Homogeneous assay

Assay format that only requires simple mix before the reading procedure

Inkjet printing

A type of computer printing that propels droplets of ink onto the substrate. A similar concept can be used to propel droplets of reactions onto the substrate

PDMS

Polydimethylsiloxane, widely used silicon-based organic polymer

Cell-free protein synthesis (CFPS)

The production of protein using biological machinery in a cell-free system

Contact printing

The printing process of a liquid directly onto the surface of a substrate

CYTOP

A fluoropolymer that dissolve in exclusive fluorine-based solvent and normally used as a thin film coating

Dipping-depositing-moving (DDM)

Dipping liquids and depositing liquids with the solid pins

Fluorescence-activated droplet sorting (FADS)

Droplets being sorted in a fluorescence-activated manner

Pneumatic valve

Component that can control the pressure, rate, and amount of air as it moves through a pneumatic system

Resazurin

A phenoxazine dye that is weakly in fluorescent, which can be reduced by NADH produced by viable cells into resorufin that is highly fluorescent

Screw valve

Rotation of the machine screw to control the opening and closing of the valve

Stroboscopic effect

A phenomenon caused by aliasing that occurs when continuous motion is represented by a series of short or instantaneous samples

Taylor-Aris disperser

A dispenser technique that can rapidly determine the diffusion coefficient of molecules