Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review

The process of choosing the most proper technique for studying the molecular interactions is based on critical factors such as instrumentation complexity, automation, experimental procedures, analysis time, consumables, and cost‐value. This review has tracked the use of affinity capillary electrophoresis (ACE) and microscale thermophoresis (MST) techniques in the evaluation of molecular binding among different molecules during the 5 years 2016–2021. ACE has proved to be an attractive technique for biomolecular characterization with high resolution efficiency where small variations in several controlling factors can much improve such efficiency compared to other analytical techniques. Meanwhile, MST has proved its higher sensitivity for smaller amounts of complex non‐purified biosamples without affecting its robustness while providing high through output. However, the main motivation to review both techniques in the proposed review was their capability to carry out all experiments without the need for immobilizing one interacting partner, besides a great flexibility in the use of buffering systems. The proposed review demonstrates the importance of both techniques in different areas of life sciences. Moreover, the recent advances in exploiting ACE and MST in other research interests have been discussed.

need for immobilizing one interacting partner, besides a great flexibility in the use of buffering systems. The proposed review demonstrates the importance of both techniques in different areas of life sciences. Moreover, the recent advances in exploiting ACE and MST in other research interests have been discussed.

K E Y W O R D S
affinity capillary electrophoresis, biomolecules, microscale thermophoresis, pharmaceuticals, protein binding

INTRODUCTION
A whole set of analytical techniques in the field of life sciences for studying the molecular interactions has been developed recently. Some of those techniques have more reliability and efficiency than others. The process of identifying the degree of interaction between two molecules is fundamental in various research areas. Drug discovery, drug design, cellular signaling, and genetic transcription are examples of such areas beside others. The probing of this interaction is the starting point to proceed with further investigations [1,2]. Choosing the proper analytical techniques to characterize the binding affinities is a critical step due to the presence of a diverse analytical toolbox being used for different interaction purposes. Therefore, the use of two analytical techniques at least is recommended and can greatly strengthen the findings. For instance, the use of molecular modeling aid with one or two experimental methods is the state of art nowadays [3][4][5][6][7][8]. Biophysical techniques are currently the leading ones in this field. That could be attributed to their fast technical developments within the last two decades, as well as the improvement of their analytical throughput and automation capabilities. The conventional fluorescence-based spectroscopy exhibited versatility in probing molecular interactions depending on recording the direct fluorescence signals or indirect quenching effect. Among fluorescence-based techniques, fluorescence correlation spectroscopy (FCS), fluorescence anisotropy (FA), and forester resonance energy transfer [9][10][11] have been most frequently used. Other spectroscopic techniques are used exclusively in identifying the dynamic structural changes, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and vibrational spectroscopic techniques [12][13][14][15][16]. Besides, surface plasmon resonance (SPR) is nowadays one of the most applied spectroscopic techniques in characterizing biomolecular interaction. SPR uses fabricated optical biosensors that sense the change in refractive index upon molecular interaction, and therefore, it is used for a wide range of interaction systems with real-time kinetic measurements [17][18][19]. On the other hand, thermodynamic characteriza-tion was preferred due to a wealth of information that can be obtained from the interactions, such as the binding constant (K a ), binding stoichiometry (n), and binding enthalpy (ΔH)/entropy (ΔS). Currently, isothermal titration calorimetry (ITC) is the predominant technique in this area [20][21][22]. Microscale thermophoresis (MST) has been introduced recently as a powerful analytical tool used for the characterization of biomolecular interactions [23][24][25][26]. Other separative analytical techniques are being used as well, with different approaches, to quantify the binding parameters. These approaches include affinity chromatography (AC), affinity capillary electrophoresis (ACE) along with conventional methods such as equilibrium dialysis, ultrafiltration, ultracentrifugation (UC), and other extraction techniques [27][28][29][30]. Figure 1 summarizes the average number of publications per year for all mentioned analytical techniques, according to the Web of Science (WOS) database, in terms of the name of the analytical technique and keywords "binding affinity." Indeed, as shown in Figure 1, the large number of publications for the AC techniques outlines its continuous developments and signifies its importance among other alternatives. However, each technique has its inherent strengths and weaknesses that should be taken in consideration, and there are many literature reviews that addressed this issue in detail [28,[31][32][33][34].
In this work for the first time, a comprehensive review of ACE and MST techniques is presented. The techniques under study somehow have certain likely similarities. First of all, both techniques depend on the molecular mobilities under directly applied forces. ACE and MST measure the binding events in free solutions without need for any further instrumental fabrications. Moreover, the reviewed techniques are probing the interactions via optical sensors; therefore, one binding partner must be optically active. Finally, the coupling between CE and MST has been easily conducted without need for instrumental modifications [35], which is promising toward devices miniaturization with several potential applicabilities in the future. This review article covers the period from the beginning of 2016 F I G U R E 1 Average number of publications/year (from January 2016 to December 2021). AC, affinity chromatography; ACE, affinity capillary electrophoresis; ED, equilibrium dialysis; FA, fluorescence anisotropy; FCS, fluorescence correlation spectroscopy; ITC, isothermal titration calorimetry; MS, mass spectrometry; MST, microscale thermophoresis; NMR, nuclear magnetic resonance; SPR, surface plasmon resonance; UC, ultracentrifugation, ultrafiltration.

F I G U R E 2
Representative electropherogram showing the principle of affinity capillary electrophoresis. Self-made figure by the corresponding author Prof. El Deeb. till the end of 2021 and provides an overview of applications where binding parameters have been quantitatively or semiquantitatively determined using either ACE-based methods or MST methods.

AFFINITY CAPILLARY ELECTROPHORESIS
ACE uses the change in electrophoretic mobility to study the noncovalent possible interactions between small molecules and different ligands [31]. ACE can be used for the characterization of interactions by determining the binding parameters (e.g., binding constants, number of binding sites, and stoichiometry). However, it can also be used to facilitate the separation of target analytes or to improve specificity and/or sensitivity of detection (i.e., sample enrichment). For instance, as shown in Figure 2, the change in the migration time of a protein as target is observed in the presence of metal as a ligand. Normalized difference of mobility ratios (ΔR/R f ) is calculated to indicate the significance of interaction. In ACE, the interacting molecules can be found free in solution or can be immobilized to a solid support. ACE modes have a broad application diversity, but it is necessary to take into account their advantages and disadvantages. Since 1997, a WOS (all databases) search on the topic of "ACE" shows that 100 and more articles had been published annually. For the period 2016-2021, this means over 600 articles had been reported. A substantial number of them were published in journals such as Electrophoresis, Journal of Chromatography A, and Analytical Chemistry ( Figure 3). Therefore, all published papers involving ACE cannot be described during this time. Basic pieces of knowledge of ACE and affinity interactions are assumed, which can be found elsewhere [31,[36][37][38][39][40][41][42][43]. This review summarizes research articles in which binding parameters are determined (quantitatively or semiquantitatively) using ACE modes in free solution in capillaries exclude microchip-analysis.
During the selected time period, a search in the WOS database under the keyword "ACE" and the following selection of articles that are focused on the determination of binding parameters, a number of 123 research articles, was identified. The search choice was focused on articles that include quantitatively or semiquantitatively determination of binding. The interactions take place in free solution without immobilization and mark of one of the interaction partners. Furthermore, chiral separations and sample enrichment were not considered in the selection.
The frequency of use of ACE modes in research articles within the period of years 2016-2021 is shown in Figure 4. The largest share of these methods is represented by ms-ACE and CE-FA. Because of the wide range of these two main approaches, the following comparison focuses on ms-ACE, CE-FA, and their variants. The review is divided into methods using mobile shifts and frontal methods. Mobility shift-based methods include conventional ms-ACE, PF-ACE, PA-ACE, and PA-PF-ACE. The approaches belonging to frontal-based methods are CE-FA, PA-CE-FA, and FACCE.

Mobility shift-based methods
ms-ACE is the most extensively used method in the measurement of affinity by CE ( Figure 4). In conventional ms-ACE, the analyte is introduced into the capillary together with an electroneutral electroosmotic flow (EOF) marker. The shift of migration time is monitored depending on the increasing concentration of interaction partners in background electrolyte (BGE). The requirement of this method is fast on and off kinetics; therefore, ligand concentrations 1-2 orders of magnitude higher than the analyte are usually used [69,70]. The most studied interactions are systems with cyclodextrins [14,45,[71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90]. They are suitable model systems for the development of new approaches in data evaluation and other theoretical models. Sázelová et al. [30]  and standard deviations of the binding constants and limiting mobilities. This study showed that, for an accurate and precise determination of the binding constants and limiting mobilities of multiply charged cyclodextrin (CD) complexes with small ions, ms-ACE was the best model for calculating BGE ionic strength. This could be attributed to the fact that the reduction in mobility related to the ionic strength would only come from the singly charged CD selectors where its charge number (Z s ) equals −1. Bertaut et al. [78] compared two approaches for the estimation of binding constants. The first approach used the migration time determined at the start of the peak, whereas the second approach aimed at determining the effective concentration corresponding to the peak apex. These two approaches allowed a correct estimation of the binding constants, even though the usual admitted requirement ([analyte] ≪ [CD]) did not comply. In addition, the new approaches allowed undertaking indirect characterization by competitive measurements and could be potentially extended to any host-guest system or any interaction study.
In 2017, PA-ms-ACE was used together with other two approaches to evaluate the interaction between loureirin B and HSA under physiological conditions (pH 7.4). The pressure method showed good repeatability with RSDs for retention times and peak areas within 2.149 and 1.228, respectively [52].
(Li + , Na + , K + , and NH 4 + ) and divalent (Mg 2+ and Ca 2+ ) cations in methanol [104], whereas in 2020, PA-ms-ACE with ICP-MS detection was used for confirmation of interaction of carboxylated core-shell magnetic nanoparticles with polymyxin B [128]. Another advance in ms-ACE was the development of MA-ACE. This approach is using moment equations to obtain not only the value of the binding constants, but also the rate constants based on the measurement of migration time and variation of the elution profile of the peaks. The factors considered in their calculations are described in more detail in a study conducted by Suzuki et al. [129]. Theoretically, through MA-ACE, it would be possible to predict the contribution of intermolecular interaction to the increase in the variance of a complex peak profile. So far, this prediction has been verified by measuring kinetic constants on model systems of cyclodextrin (β-CD) with 2-phenoxypropionic acid [62] and sulfated-β-cyclodextrin with thymol [130]. These results suggest that MA-ACE could be an effective tool for future kinetic studies of intermolecular interactions. These results suggest that MA-ACE could be an effective tool for future kinetic studies of intermolecular interactions that can be deter-mined without immobilization and labeling one of the molecules.
For more accurate results, the ms-ACE method can be combined with an ionic liquid-based aqueous twophase system (ILATPS/ACE). In 2018, the ILATPS/ACE system used 1-butyl-3-methylimidazolium chloride to rapidly and precisely estimate the binding constants of anticancer drugs methotrexate (MTX) and vinblastine (VBL) with human alpha (α1)-acid glycoprotein (AGP) [131]. The second ILATPS/ACE system using 1-ethyl-3-methylimidazolium chloride was used to determine the binding between AGP with the anticancer drugs chlorambucil (CHL) and dacarbazine (DAC) [132]. The obtained results had been in good agreement with results obtained by high-performance affinity chromatography. In addition, the ILATPS/ACE systems proved being suitable for the binding analysis of proteins with a variety of acidic or basic drugs.
Another approach that is included in mobility-based methods is PF-ACE. This is a variant from the conventional ms-ACE. The main difference between them is that the capillary is not filled with ligand-containing buffer in the whole its length [133][134][135]. The ligand-containing buffer only fills in part of the capillary. Therefore, it creates a ligand-plug in the capillary which can have the range from 0% to 100% of the effective length, and the residue of the capillary is filled with pure buffer solution. Compared to conventional ms-ACE, where the inlet and outlet vials are filled with a ligand-containing buffer during analysis, in PF-ACE, there is only pure buffer in the vials [50]. During the selected period, PF-ACE was mainly used to characterize protein-ligand interaction [51,[136][137][138][139]. Farcaş et al. [139] developed a partial-filling electrophoretic mobility shift displacement assay. This indirect approach can be applied for negatively, positively charged, and neutral molecules, as well as molecules that do not have absorbances in the UV-VIS range, for which the determination has been problematic so far.
Another newly created modification is the PA-PF-ACE method [140]. This method was applied for the determination of the apparent binding constants of human insulin (HI) hexamer complexes with serotonin, dopamine, arginine, and phenol. In this study, the pressure was applied against the direction of the EOF, and this setup ensured that HI migrates back to the anode, whereas the cationic ligands (serotonin, dopamine, and arginine) and anionic ligand (phenol) migrated through the plug of HI to the detector.
In 2019, the approach of flow-through partial-filling ACE (FTPFACE) was published by Hutanu et al. [141]. This method had been established for the determination of mixture of two similar monoclonal antibodies (mAbs). The addition of a specific ligand resulted in the complexation of one mAb in the co-formulation, thus changing its migration time through the electric field. That allowed the characterization of the charged variants of the non-shifted mAb without interferences. The presented FTPFACE approach requires only very small amounts of ligands and provides complete comparability with a standard CZE of a single mAb [141]. Table 2

Frontal-based methods
Frontal methods differ from other ACE methods by the amount of sample injected into the capillary. Due to the long plug injection, the CE-FA electropherogram does not contain classical Gaussian-shaped peaks but rectangular plateaus. All CE frontal methods require two series of measurements, one series of pre-equilibrated mixtures with a fixed amount of analyte and several ligand concentrations and the other series with pure ligand in different concentrations to set up a calibration curve. In contrast to ms-ACE, the binding stoichiometry can also be determined [68,144]. Applications in the period 2016-2021 for CE-FA methods are very diverse. Most often, we encounter the determination of binding parameters between proteins and drugs [47,52,57,[145][146][147][148]. However, CE-FA was used to characterize the interactions of glycosaminoglycans-protein [149], macrophage-drug [150], heme-insulin [151], platelets-small molecules [152], CD-small molecules [153], ss-DNA-small molecules [49], antigen-adjuvant [58], polymer-copolymer [59,60,154], and protein-polysaccharide [55]. Nevídalová et al. presented the possibility of using the CE-FA method for displacement studies [147]. Recently the same group presented the possibility of combining the CE-FA method with online sample mixing directly in the capillary [148].
External pressure can be applied during separation to speed up CE-FA analyses. This method was used several times during the studied period. In 2018, it has been found that a typical CE-FA setting will not be affected when an external pressure of less than 5 psi is used [153]. In 2020, Yang et al. characterized interaction between Bcl-2 oncogene promoter I-Motif DNA and flavonoids. Following year, the same group used PF-CE-FA method for the investigation of interaction between the human oncogene c-KIT promoter G4 and natural binders [155].
The continuous variant of CE-FA is FACCE. As a conventional CE-FA, it provides information on the binding constant and stoichiometry. In both methods, a long zone of the sample is dosed into the capillary, and the difference between them occurs only after injection of the sample. In the classic CE-FA, the inlet and outlet vials are filled only with pure buffer, whereas in FACCE, the inlet vial is filled with the injected sample, and the outlet vial is filled with pure buffer. The interactions between model polyanions and polycations were studied in 2017 and 2018 [58,59,154]. In 2020, an interesting application of FACCE was published, which reported a method that was used to study the interactions of antigen and adjuvant in vaccine products. The binding parameters between the anionic polymeric adjuvant (polyacrylic acid, SPA09) and the cationic vaccine antigen under development for the treatment of Staphylococcus aureus were determined. It has been found a strong dependence of the binding parameters with the ionic strength and also that the concentration of polymeric adjuvant significantly modifies the ionic strength of the formulation, the extent of which can be estimated and corrected [58]. The applications of CE-FA [47,52,55,57,[145][146][147][148][149][150][151][152][153][155][156][157][158][159][160][161] and FACCE [58][59][60]154] are summarized in Table 3.

MICROSCALE THERMOPHORESIS
MST is a biophysical technique that gained popularity after being introduced to the scientific realm at the beginning of this millennium. Note: by the German scientist Ludwig; however, the Swiss scientist Soret deliberated mathematically in detail [162,163]. In MST, the samples are placed in µM concentrations into thin capillaries. An IR laser beam is focused on the capillaries center in order to generate a temperature gradient ( Figure 5). The thermophoretic signal is measured using fluorescence detector that measures the fluorescence at that spot. Any escaping fluorescent molecules under the thermal gradient would result in a drop in the thermophoretic signal. In such a way, if a molecule is bound to a certain ligand, this would affect the speed at which it escape from the temperature gradient and hence affect the thermophoretic signal [26]. This variation in fluorescent signal created upon the heating of the sample is hence dependent on the binding and is known as temperature related intensity change (TRIC) [164]. The chemical environment of the fluorescent probe directly affects the TRIC, where it could be altered by any binding events with a possible ligand [164]. Historically for decades, thermophoresis has been exploited earlier for gas mixtures, inorganic chemistry, organic polymers, and several physical experiments related to the space and microgravity environments [163]. During the last two decades, there had been numerous attempts to investigate the thermophoresis of biomolecules such as DNA, Proteins, and enzymes [165][166][167][168][169]. Braun et al. are the pioneers for an innovative project for investigating thermal diffusion optically as a fluorescence intensity for biomolecules in microfluidic environments [ 166,167,170,171]. Theoretically, thermophoresis can be defined as a Soret coefficient of the free molecules in solutions according to the following equation [172]: where S T is the Soret coefficient, A is the surface area of the molecules, σ eff is the effective charge, ∆S hyd is the hydration shell effect, λ DH is the Debye-Hückel screening length, ɛ is the dielectric constant, and β is temperature derivative of ɛ. Accordingly, MST is sensitive to the minor changes in molecular size, hydration entropy, and effective charges upon molecular/biomolecular interactions. Generally, MST has been invented recently to fill some gaps available in the competing biophysical techniques such as SPR, ITC, and several spectroscopic techniques [173]. In comparison to SPR, MST is capable of measuring the binding events in free solution directly without immobilizing fabrication. One of the main disadvantages of MST, though being a sensitive sensor, is its low capabilities to measure the binding kinetics in a steady equilibrium state, which can be considered the main advantage of SPR technique [26]. However, recent studies showed MST capability to measure the binding kinetics in dynamic way such as introduced by Jerabek-Willemsen et al. [174], who demonstrated MST ability in quantifying enzyme kinetic.
Recently, Stein et al. [175] proved the cost-effectiveness of MST in measurement of reaction kinetics and introduced a new term known as kinetic microscale thermophoresis (KMST). Slight modifications were made to the conventional MST hardware for increasing the thermal dissipation of a sample ( Figure 6). This in turn would be beneficial in deducing the kinetic relaxation from the fluorescent intensity and was successfully applied in studying the kinetic parameters of DNA hybridization [175]. KMST has hence proved both time and cost-effectiveness over SPR, where the reaction kinetics and K a can be both measured in a single experiment. However, there are many drawbacks of SPR artifact such as binding overestimation and concentration depletion [173,174,176]. Moreover, MST has a wider flexibility in selecting the working buffers in comparison to ITC which is restricted to certain buffers with low enthalpy ionization. Although ITC gives affinity, stoichiometry, and thermodynamic parameters in free solution and label free system, but it suffers from low sensitivity, slow throughput, time consumption, and utilization of higher amounts of samples to obtain sufficient heat signals [174,177].
FA and FCS are fluorescence-based methods and similar to MST. However, FCS technique depends on the change in diffusion time of fluorescent molecules through the detecting of fluorescence fluctuation upon the diffused fluorescent molecules out the focused volume. FA almost has the same principle, except that it measures the change in rotational diffusion time. Thereby, polarized time, which is the time interval between absorption and emission molecular orientation, has been used to calculate the binding or interaction. Both, FA and FCS, techniques have the drawbacks of being time-consuming, and there is a need to perform more optimization in comparison to other biophysical techniques [178][179][180][181].
Previously, our research group tracked the MST experimental methods till the beginning of 2017 [182]. During 2016-2021, MST-based methods for estimating the binding events had been increasing yearly. According to scientific platforms such as WOS and Scopus, there are more than 600 research articles have been published, of which 50 articles have been selected according to the research area, the number of citations, and new trends in the relevant research articles.

MST in drug discovery and drug design
During the last decade, MST has been extensively used in drug design and drug discovery. Among those important discoveries, those biomolecules targeted against tumors, and antivirals gained much attention. Therefore, a considerable attention for those biomolecules can be expressed by the number of scientific publications. Protein kinase family is one of the most targeted proteins. Linke et al. [183] published an interesting work using automated MST for screening of lead compounds depend on fragments-based lead discovery. Around 193 fragments had been tested against mitogen/extracellular signal regulated kinase (MEK1) which have an important signaling role in cancer, whereas 73 fragments had been bound to MEK1, and 120 fragments did not show binding affinities. Furthermore, the obtained data were compared with other findings from competitive biophysical techniques such as SPR and dynamic force spectroscopy and were confirmed by X-ray crystallography. The results confirmed the superiority of MST in quantitation of the large number of hits in comparison to SPR and DSF. Davis et al. [184] discovered a small molecule (ML364) as a potent inhibitor for deubiquitinase (USP2) protein that has a regulatory role in cell cycle and upregulated in various cancer cells. MST method was developed to investigate the inhibitory effect of ML364 toward USP2 with estimated K d = 5.2 µM. Furthermore, biochemical assays in vitro to different cell lines with estimated IC50 = 1.1 µM. In the same manner, Bao et al. [185] targeted Hexokinase 2 with 13 natural inhibitors using MST that was confirmed by cell-based assays. Therein, there were various natural small molecules that showed a promising inhibitory effect to different proteins kinases using MST as a choice analytical technique [186].
Recently, Fischer et al. [187] discovered that new inhibitors based on in silico drug screening for about 2556 750 lead compounds via using of ZINC database and molecular docking with certain criteria. Five leads have been selected to study the inhibition effect against translationally controlled tumor protein (TCTP). TCTP is an interesting targeted protein that has a vital role in cell growth and overexpressed in cancer cells. MST was used to confirm in silico results. Another promising study was conducted by McPherson et al. [188]. They designed nine phenazopyridine derivatives (PAP) and identified as potent inhibitors for translation synthesis, which is the important pathway for DNA polymerase to resist the platinating chemotherapeutics such as cisplatin. MST was used to probe the interaction between Rev1-CT protein and the designed (PAP) derivatives. The binding events were estimated as K d that exhibited a strong binding affinity in low µM ranges and similar to the estimated binding events for Rev1 to polymerase (Pol k ) that obtained by SPR. Additional MST applications are listed in Table 4.
MST applications for testing new antiviral agents during 2016-2021 represent one of the main research areas in drug discovery and design. Nowadays, it has been paid more attention as a hectic topic due to breakthrough of the recent pandemic of SARS coronaviruses (SARS-CoV-2). Repurposing of existed antiviral drugs opened new prospects for fast screening and shortened the time to find new drug molecules. Herein, Puhl et al. tested Ebola and Marburg virus inhibitors against SARS-CoV-2 virus [189]. Three antiviral drugs have been selected in this study, including pyronaridine, tilorone, and quinacrine, and were tested against various cell lines infected with SARS-CoV-2 virus. MST was used to investigate the binding of these molecules with the spike proteins, and K d values were estimated TA B L E 4 Microscale thermophoresis (MST) data of different binding applications.

Application
Binding system Binding parameters

Confirmatory techniques
Ref.

MST applications in drug discovery and development
Lead discovery against MEK I kinase Small molecules-protein interaction K d = 4.5 µM in absence of MgCl 2 , 140 µM in presence of MgCl 2 X-ray crystallography [183] Drug discovery, protease inhibitors (USP2) Small molecules-aptamer interaction Biochemical assay [184] Inhibitory effect of small molecules to Hexokinase 2 Small molecule-protein interaction Molecular docking Biochemical assays [185] Inhibitory effect of small natural molecule (Esculetin) to tumor cell glycolysis Small molecule-protein interaction  [190] Drug discovery against (SARS-CoV-2) Molecular docking [191] Drug discovery against (SARS-CoV-2) Peptides-protein interaction K d = 5.4 µM Molecular docking [192] Drug discovery, ischemic heart disease treatment Small molecules-protein interaction in hundreds nanomolar scale for tilorone and pyronaridine, whereas quinacrine did not show inhibition activity against SARS-CoV-2 spike receptor protein.
Another research strategy for MST is focused on the screening for new low molecular weight peptide candidates that could have the ability to reduce immune response and possess antibody similar effect [190]. Some peptides have been investigated for blocking or interrupting the protein-protein interaction of virus spike protein with membrane angiotensin-converting enzyme-2 receptor (ACE-2) that was recognized as a potential pathway for viruses to cell entry. Sadremomtaz et al. [190] tested six synthetic peptides using molecular docking, and then the results were confirmed with MST experiments. MST results found that two of six candidate peptides exhibited higher affinity to ACE-2 receptors with estimated K d values ∼10 nM. Concomitantly, the same approach has been carried out by Odolczyk et al. [191], where MST results revealed the strong binding affinity of two peptides toward receptor binding domain of SARS-CoV-2 spike protein (RBD-S) with estimated K d 210 and 280 nM. Another approach carried out by Gao and Zhu [192], where the fungal defensin had been chosen based on antimicrobial activity and the reported roles in blocking virus-cell membrane interfacing. Micasin was bound significantly with RBD as predicted with molecular docking and confirmed with MST. However, K d value was estimated in low micromolar range (∼2 µM) which is slightly higher than other peptide candidates. Other featured MST applications for antiviral activity [23, rather than SARS-CoV-2 are summarized in Table 4.
Moreover, MST other than the aforementioned applications, which represent the predominant ones in 2016-2021 with a large number of featured publications, there is some featured research that was conducted with different targets. For instance, Wang et al. [193] identified natural cyclopeptide known as destruxin A5 to act as a potent blocker for signaling of surface receptor tyrosine kinases (PDGF-BB/PDGFR-ββ) that might be effective in attenuating liver fibrosis. In that study, the researchers used computational analysis aided by biophysical techniques such as MST, SPR, and thermal shift technology to quantify the binding events of destruxin A5 with extracellular domain of human PDGFR-β, and the estimated K d values were inconsistent for MST and SPR techniques (∼5 µM). Another study was conducted by Zhong et al. to investigate the role of salvianolic acid A (SAA) as a cardioprotective agent, which is an active compound present in known herbal medicine called Danshen [194]. After several evidences that support the benefits of this medicinal plant in the cardiac events, the researchers tried to identify a new target for SAA using SPR and MST. The estimated binding events were in accordance for both techniques between SAA and transgelin that identified as a new target for the first time.

MST in biosensors development
Biosensor technology is a pioneering method of analysis recently due to its high selectivity and rapid detection.
Biosensor-based devices open new prospects to overcome the challenges related to the existed conventional techniques [195,196]. MST had been involved in biosensor development via exploiting of thermal diffusion properties of the analytes upon binding. Aptasensors are the main developed biosensors that had been tested via MST. Valen-zano et al. [229] screened and identified DNA aptamers for tyramine as a targeted toxic biogenic amine presents mainly in fermented food beverages. Around 15 aptamers candidates were successfully isolated and selected to investigate their binding against small molecule tyramine. The binding events were estimated as K d in the range of 0.2-153.0 µM. Tyr-10 and Tyr-14 were the promising aptamers and recommended to be used as aptasensors for tyramine in food and beverages. On the other hand, impedimetric aptasensor was presented by Reich et al. for the detection of S. aureus [196]. The aptamer was immobilized to the gold electrode that exhibited a selective binding to S. aureus surface protein A. MST was used to investigate the binding affinity between aptamer S. aureus targeted proteins that exhibited a strong binding affinity with K d in low nanomolar ranges (16-22 nM). Similarly, Prante et al. [230] developed a new biosensor targeting 25-hydroxyvitamin D by using of aptamer-based assay, and MST has been used to investigate the binding affinity of aptamer toward 25-hydroxyvitamin D. The binding event in that study was estimated as K d values of ∼15 nM which exhibited a strong binding between the two partners. Those results were in accordance with the previous reported methods using ITC (K d = 11 nM). Recently, Yang et al. [197] interestingly tested the affinity of a bifunctional aptamer against mycotoxins Zearalenone and ochratoxin simultaneously using aptamer-based MST aptasensor. The developed MST aptasensor was applied to determine the aforementioned mycotoxins in corn oil with estimated limit of detection of 0.12 nM and recoveries of 93.31%-104.19%. One further step in probing of toxins in environment, Mukherjee et al. [198] investigated DNA aptamer to detect marine biotoxin known as 20-methyl spirolide G in seafood. MST-based aptasensor indicated a linear detection range from 1.9 to 125 000 pg/mL and the recoveries in the range of 86%-108%. Furthermore, MST exhibited a powerful sensing method for inorganic phosphate (Pi). Franz et al. [195] developed MST method for detecting and quantifying free Pi during enzymatic reaction in micro-milliliter sample volumes (∼10 µL) for the first time. This interesting work was carried out via investigating the entropy-driven thermodiffusion of a protein-based Pi-sensor to enzyme-catalyzed nucleotide hydrolysis reactions, thereby, allowing real-time quantification of Pi in various ATPas activity reactions. Further, MST featured applications are listed in Table 4.

MST in cell biology
Studying of cell biology is an essential step to understand the physiological/pathophysiological processes. The cellular decisions such as cell growth and division, differentiation, and apoptosis required for the functionality of multicellular organisms. Furthermore, cell signaling is important and identifying the abnormal cell signaling pathways are early warning of many disease state such as cancer, autoimmune diseases, and so on [231]. MST as a tool for the characterization of biomolecular interaction was invested greatly from the early stage of MST technique infancy. During 2016-2021, there were plenty of featured research articles in this area. Lippok et al. [199] investigated the von Willebrand factor dimerization mechanism to answer the question of potential protein catalysis. They used a set of biophysical techniques, including high-resolution stochastic optical reconstruction microscopy, atomic force microscopy, MST, and FCS. The binding events were estimated as K d values of 5 236 and 282 nM by MST and FCS, respectively. Another work by Heintz and Schlichting [200] used MST and another biophysical techniques to investigate the structural changes that blue light causes in the Aureochrome1a photoreceptor found in the algae. MST estimated of the monomer dimerization as K d of 13.6 ± 1.4 µM. In the same period, one featured work for Uzarska et al. [232], by using MST and other biophysical techniques to study the role of mitochondrial BOLA family proteins especially (BOl1 and Bol3) as assembly factors in iron-sulfur proteins biogenesis. MST, circular dichroism, NMR, and size exclusion chromatography were used to characterize the binding affinity of BOLA proteins with different cofactors. MST showed a specific interaction between the two BOLA proteins and both apo-and holo-NFU1 (protein coding gene). The estimated K d values of the interactions were in the range of 3 µM, except a fourfold higher affinity detected for BOLA3 and holo-NFU1 (K d = 0.8 mM). They concluded that Bol3 mutations might be expected in patients. Another work related to cell biology and cell transportation was carried out by Heybrock et al. [201], using molecular modeling, crosslinking studies, MST, and cell-based assays to study the role of lysosomal integral membrane protein type 2 (LIMP-2) in cholesterol transport. MST data exhibited direct interactions between LIMP-2 protein and cholesterol, and the binding events were estimated as EC50 of 112 nM which indicate the strong affinity between the two partners. The results supported a fundamental physiological role of LIMP-2 in lipid transport and may help in drug development for LIMP-2 protein as a targeted protein. One interesting research by Deng et al. [202] was published for the investigation of Streptococcal B pathogenesis mechanism in meningitis. The work group determined that BspC (antigen I/II protein) interacts with the host cytoskeleton component vimentin, and this interaction was confirmed by various methods, including bacterial two-hybrid assay, MST, immunofluorescent staining, and imaging flow cytometry. MST data showed the direct interactions between BspC and vimentin with estimated binding constant K d of 3.39 µM which is similar to the data obtained by other methods. Recently, Alba et al. [203] recognized a new targeted protein (NEDD9) as a mediator of platelet-endothelial adhesion in pulmonary circulation. That interesting research explains the molecular mechanism of hypoxia signaling pathways. Furthermore, NEDD9 antibodies were developed and tested to investigate the inhibitory effect to protein-protein interaction. MST data exhibited strong binding between NEDD9 and P-selectin ligand and the estimated K d of 13.9 nM. Another interesting contribution during Covid-19 pandemic by Zinzula et al. [204] investigated the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD) via using of numerous analytical techniques such as size exclusion chromatography and UC. MST experiments demonstrated the interaction between SARS-CoV-2 (CTD) and ssRNA oligomer at micromolar affinity (K d = 4 µM). Further, MST applications are summarized in Table 4.

CONCLUDING REMARKS
A hundred of published articles that were based on ACE or MST techniques during the period 2016-2021 were reviewed. Although the use of ACE could be limited by some repeatability concerns related to its injection volume precision, the cost-effectiveness, fast analysis time, low consumption of used samples, and operation simplicity are positive advantages. Moreover, the specificity of ACE and its detection sensitivity is much improved by the enhanced separation of the targeted analytes. The mechanism of operation of ACE technique makes it the best in the study of the amphoteric protein-based biomolecules such as DNA, RNA, and genetic sequencing. On the other hand, although MST is intruded by some interferences due to the presence of high number of aromatic rings within most of the studied proteins, especially for nonfluorescent or weakly fluorescent target molecules; however, the capability of labeling such target molecules can easily overcome this limitation. MST can easily be setup and optimized, where precise data are acquired in short time and at high quality. On the top of that, the KMST has enhanced the time/cost-effectiveness of MST by slight modifications in MST hardware to measure both K a and kinetics in a single practice. MST is powerful when it comes to complex matrices and picomolar concentrations of biomolecules owing to its thermophoretic fluorescent mechanism. Based on such review, a great possibility was found to exploit these two techniques comprehensively in estimating the interactions among biomolecules, small molecules, and even metal ions. Another added strength point is their low sample consumption. On the other hand, the simultaneous adaptation of both techniques is a promising option to enhance their efficiency in estimating the binding parameters between varying types of interacting molecules. This hypothesis can be proved by a recently first to be published coupling of CE and MST for dynamic equilibrium analysis [233], which opens the horizons for adapting a novel ACE-MST interface. Moreover, the current software and computational developments furnish the way for a full automation of both techniques within the next few years. Open access funding enabled and organized by Projekt DEAL.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have declared no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All manuscript data are available from the corresponding author upon request.