Development of the Sensing Platform for Protein Tyrosine Kinase Activity

biosensors

Article Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Lan-Yi Wei 1, Wei Lin 1, Bey-Fen Leo 2,3 , Lik-Voon Kiew 1,3,4, Chia-Ching Chang 1,3,5,6,7 and Chiun-Jye Yuan 1,3,5,8,*

1 Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu 30068, Taiwan; [email protected] (L.-Y.W.); [email protected] (W.L.); [email protected] (L.-V.K.); [email protected] (C.-C.C.) 2 Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia; [email protected] 3 Taiwan-Malaysia Semiconductor and Biomedical Oversea Science and Technology Innovation Center, National Yang Ming Chiao Tung University, Hsinchu 30068, Taiwan 4 Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia 5 Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B), National Yang Ming Chiao Tung University, Hsinchu 30068, Taiwan 6 Department of Electrophysics, National Yang Ming Chiao Tung University, Hsinchu 30010, Taiwan 7 Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan 8 Institute of Molecular Medicine and Bioengineering, National Yang Ming Chiao Tung University, Hsinchu 30068, Taiwan * Correspondence: [email protected]; Tel.: +886-3-573-1735

Abstract: A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 µL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of Citation: Wei, L.-Y.; Lin, W.; Leo, 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the B.-F.; Kiew, L.-V.; Chang, C.-C.; Yuan, C.-J. Development of the Sensing inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition Platform for Protein Tyrosine Kinase of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the Activity. Biosensors 2021, 11, 240. significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results https://doi.org/10.3390/ suggest the potential of the developed miniature sensing platform as an effective tool for detecting bios11070240 different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases. Received: 20 May 2021 Accepted: 13 July 2021 Keywords: biosensor; electrochemical analysis; tyrosine kinase Published: 15 July 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- Protein tyrosine kinases are one of the phosphotransferase families that transfer γ- iations. phosphate of adenosine triphosphate (ATP) to the tyrosine residues of the target proteins [1]. Tyrosine phosphorylation has been shown to play essential roles in many cellular events, such as cell proliferation and differentiation, protein synthesis, cell cycle, embryo development, cell migration and apoptosis [2–5]. Dysregulation of protein tyrosine kinases, either Copyright: © 2021 by the authors. by overexpression or overactivation, leads to many diseases, such as diabetes, neuronal Licensee MDPI, Basel, Switzerland. degenerative diseases, and cancers [6–12]. For example, many human tumors, such as This article is an open access article non-small cell lung cancer, squamous cell carcinoma of the head and neck, glioblastoma, distributed under the terms and pancreatic cancer, ovarian cancer, breast cancer, and prostate cancer were found to closely conditions of the Creative Commons Attribution (CC BY) license (https:// relate to the over-activation of human epidermal growth factor receptor 2 (Her2), a sub- creativecommons.org/licenses/by/ family of ErbB (erythroblastic oncogene B) protein tyrosine kinases [8,10–15]. Meanwhile, 4.0/). dysregulation of hematopoietic cell kinase (Hck), a Src family protein tyrosine kinase [16],

Biosensors 2021, 11, 240. https://doi.org/10.3390/bios11070240 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, 240 2 of 11

was found to associate with many human diseases, including cancers, autoimmune diseases, and inflammation [17]. Hence, it is essential to determine the activity of tyrosine kinases to reveal the development and progression of diseases and to understand the molecular mechanisms leading to the diseases. To detect the activity of protein tyrosine kinases, several direct methods, i.e., 32P- phosphate labeling [18], fluorescence-labeling [19] and mass spectrometry analysis [20], and indirect methods, i.e., detection with a phosphoryl tyrosine-specific antibody [21] and the quantum dot-based method [22], were developed and widely used. However, these conventional methods exhibit different disadvantages. For example, safety is the major concern of using radio isotope labeling; while the detection of protein phosphorylation by indirect methods, i.e., a phosphoryl tyrosine-specific antibody and the quantum dot-based method, suffered from high reagent cost and complicated detection procedures. Although a trace phosphorylation of residues can be sensitively detected on the mass spectrometer, the expansive instrumentation blocks its extensive usage by scientists. Hence, a rapid, simple, and effective method for the detection of protein tyrosine kinase activity in vitro would be favorable for clinical diagnosis, prognosis of drug treatment, and drug design and screening. Electrochemical methods have been developed for the detection of interactions be- tween protein–protein [23–25], protein–DNA [26] and enzymatic reactions, including the protein tyrosine kinases activity [27–34]. These methods can be categorized into label-based and label-free methods, based on the sources of redox responses. The label-based methods are mainly based on the labeling of the electro-inactive phosphate group with electroactive species, such as ferrocene-conjugated ATP [28–30] and catalytic components, such as gold nanoparticles and redox enzyme [31–34], whereas the label-free methods are based on the direct oxidation of tyrosine residues with or without amplifications [35–38]. Although both methods exhibited high sensitivity to detect tyrosine phosphorylation, the label-free methods are more direct without additional steps of labeling. Recently, a novel label-free protein tyrosine kinase biosensor was reported by oxidizing the tyrosine residue(s) on the substrates of protein tyrosine kinases by tyrosinase to generate L-DOPA quinone, which was then reduced on the electrode to give a reductive response. The tyrosinase-mediated tyrosine oxidation can be blocked by the phosphorylation and suppress the reductive responses [38]. Thus, the tyrosine kinase activity can be revealed on the biosensor by the decrease in the reductive responses of their substrates. This result suggests that this biosensor can be utilized to detect the activity of various protein tyrosine kinases. In this study, a tyrosinase-based miniature protein tyrosine kinase sensing platform was developed for the quick detection of the activity of various protein tyrosine kinases, such Hck and Her2, in a small sample volume.

2. Materials and Methods 2.1. Materials Carbon ﬁber paper (CFP, MGL190) was purchased from AvCarb (Lowell, MI, USA). Tyrosinase, CP-724714, Src inhibitor-1 (src-I1), HEPES, chitosan, N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (Darmstadt, Germany). Hck and Her2 were bought from SignalChem (BC, Canada). ELMO-Y511 (QNLSYTEIL, a Hck peptide substrate) [39], FLT3 (DNEYFYV, a Her2 peptide substrate) [29], c-Src substrate-1 (YIYGSFK, a c-Src peptide substrate) [38], and kemptide (LRRASLG, a PKA peptide substrate) were synthesized by AngeneBiotech (Taipei, Taiwan). MgCl2 was purchased from Yakuri Pure Chemical Co. (Kyoto, Japan). Sodium dihydrogen phosphate, sodium hydrogen phosphate and MnCl2 were supplied by SHOWA (Kumamoto, Japan). DMEM (Dulbecco’s Modiﬁed Eagle’s medium) and fetal bovine serum were obtained from HyClone Laboratory Inc. (Marlborough, MA, USA). Biosensors 2021, 11, 240 3 of 11

2.2. Preparation of Tyrosinase-Based Electrode The working area (0.3 × 0.3 cm2) of a CFP strip (0.3 × 1.0 cm2) was ﬁrst cleaned with oxygen plasma using a plasma cleaner (Atto, Diener Electronic, Ebhausen, Germany). The plasma treatment was performed under the oxygen pressure of 0.4 N and a power

Biosensors 2021, 11, x FOR PEER REVIEWof 75 watts for 15 s. After plasma treatment, the CFP strip was rinsed3 of 11 once with double

deionized water (d.d. H2O), followed by spreading 2 µL of 0.4% chitosan/1% acetic acid solution on the working area and then air-dried. The immobilization of tyrosinase (2002.2. Preparation U) was performedof Tyrosinase-Based by first Electrode mixing with the mixture of 10 mM NHS and 10 mM EDC in 50The mM working phosphate area (0.3 buffer, × 0.3 cm pH2) of 6.6. a CFP After strip incubating(0.3 × 1.0 cm2) at was room first cleaned temperature with for 15 min, the tyrosinase/EDC/NHSoxygen plasma using a plasma mixture cleaner (Atto, (6 µL) Diener was Electronic, then spread Ebhausen, on the Germany). working The area of CFP under theplasma room treatment temperature was performed for at under least the 2 h. oxygen pressure of 0.4 N and a power of 75 watts for 15 s. After plasma treatment, the CFP strip was rinsed once with double deionized water (d.d. H2O), followed by spreading 2 μL of 0.4% chitosan/1 % acetic acid solu- 2.3. Fabrication of the Miniature Protein Tyrosine Kinase Sensing Platform tion on the working area and then air-dried. The immobilization of tyrosinase (200 U) was performedThe miniatureby first mixing tyrosine with the kinasemixture sensingof 10 mM platformNHS and 10 consists mM EDC of in a50 polylactic mM acid (PLA) holder,phosphate a workingbuffer, pH electrode,6.6. After incubating a platinum at room counter temperature electrode for 15 andmin, athe Ag/AgCl tyrosi- reference elec- nase/EDC/NHS mixture (6 μL) was then spread on the working area of CFP under the trode (Figure S1, Supplemental information). The PLA holder, generated by the 3D printer room temperature for at least 2 h. (Botfeeder Co., Taiwan), is a rectangular block (4.2 × 4.2 × 7.5 cm3) with a cavity of 1.82.3. ×Fabrication1.9 × 5.7 of the cm Miniature3. It is composedProtein Tyrosine of Kinase two parts: Sensing (a) Platform the desk-like top part (~5.5 cm height) containsThe miniature a hole oftyrosine 0.8 cm kinase in diametersensing platform to place consists the of reference a polylactic electrode; acid (PLA) (b) the C-shaped bottomholder, a partworking (~2 electrode, cm height) a platinum contains counter a rectangular electrode and cleft a Ag/AgCl of 0.3 ×reference0.05 cm elec-2 to fix the working electrode.trode (Figure Once S1, Supplemental the holder information). was assembled, The PLA theholder, counter generated (Pt by wire) the 3D and printer reference electrodes (Botfeeder Co., Taiwan), is a rectangular block (4.2 × 4.2 × 7.5 cm3) with a cavity of 1.8 × (CH1.9 × 5.7 Instruments, cm3. It is composed West Lafayette,of two parts: IN, (a) the USA) desk-like were top fixed part on (~5.5 top cm of height) the working con- electrode at a verticaltains a hole distance of 0.8 cm ofin diameter 2 mm (Figure to place S1).the reference electrode; (b) the C-shaped bottom part (~2 cm height) contains a rectangular cleft of 0.3 × 0.05 cm2 to fix the working elec- 2.4.trode. Protein Once the Tyrosine holder was Kinase assembled, Reaction the counter (Pt wire) and reference electrodes (CH Instruments, West Lafayette, IN, USA) were fixed on top of the working electrode at a µ verticalThe distance kinase of 2 reaction mm (Figure of HckS1). and Her2 was performed by mixing 2 L of protein kinase stock solution with 18 µL kinase reaction mixture (60 mM HEPES, pH 7.5, containing 3 mM MgCl2.4. Protein2, 3 mMTyrosine MnCl Kinase2, 100ReactionµM substrate peptide and 0.5 mM ATP) in a microfuge tube and incubatedThe kinase at 30reaction◦C for of Hck a period and Her2 of was time. performed by mixing 2 μL of protein kinase stock solution with 18 μL kinase reaction mixture (60 mM HEPES, pH 7.5, containing 3 2.5.mM ElectrochemicalMgCl2, 3 mM MnCl Measurement2, 100 μM substrate peptide and 0.5 mM ATP) in a microfuge tube and incubated at 30 °C for a period of time. The electrochemical responses of the tyrosine kinases on the miniature sensing plat- form2.5. Electrochemical were determined Measurement by amperometric current–time responses (i–t curve). The space betweenThe electrochemical working and responses counter/reference of the tyrosine kinases electrodes on the (Figureminiature1 )sensing was first plat- filled with 18 µL ofform 100 were mM determined phosphate by amperometric buffer (pH current–time 6.5). The responses electrochemical (i–t curve). The measurement space be- was started tween working and µcounter/reference electrodes (Figure 1) was first filled with 18 μL of by100 injectingmM phosphate 1–2 bufferL peptide (pH 6.5). stock The elec solutiontrochemical of kinasemeasurement reaction was started mixture by in- into the phosphate bufferjecting 1–2 with μL peptide pipetting stock and solution monitoring of kinase reaction the electrochemical mixture into the phosphate responses buffer under the potential ofwith− 0.2pipetting V vs. and Ag/AgCl monitoring [38 the] onelectroc thehemical electrochemical responses under analyzer the potential CHI 6116e of −0.2 (CH Instruments, WestV vs. Ag/AgCl Lafayette, [38] IN,on the USA). electrochemical Kinase activity analyzer ofCHI Hck 6116e and (CH Her2 Instruments, was determined West by the net electrochemicalLafayette, IN, USA). responses Kinase activity before of Hck and and after Her2 phosphorylation. was determined by the net electrochemical responses before and after phosphorylation.

Figure 1. Close up view of the miniature protein tyrosine kinase sensing platform. The organization of working, counter and reference electrodes in the platform was revealed in the picture (Left) and the diagram (Right). Biosensors 2021, 11, x FOR PEER REVIEW 4 of 11

Figure 1. Close up view of the miniature protein tyrosine kinase sensing platform. The organiza- Biosensors 2021, 11, 240 tion of working, counter and reference electrodes in the platform was revealed in the picture (Left4 of 11) and the diagram (Right).

3. Results and Discussion 3. Results and Discussion 3.1.3.1. Characterization Characterization of of Mi Miniatureniature Detection Detection Platform Platform AA miniature miniature protein protein tyrosine tyrosine kinase kinase sensing sensing platform platform (Figures (Figure1 1 andand FigureS1) with S1) a withthree- a electrodethree-electrode setup was setup developed was developed that allowed that allowed the detection the detection of peptide of peptide substrates substrates in a vol- in a umevolume as small as small as 18 as μ 18L µ(FigureL (Figure 1).1 The). The ability ability of of the the miniature miniature sensing sensing platform platform to to detect detect thethe peptides peptides with with tyrosine tyrosine residue(s) residue(s) was was de demonstratedmonstrated byby measuringmeasuring thethe i–ti–t responsesresponses of of c- c-SrcSrc substrate substrate 1 1 peptide, peptide, which which was was successively successively added added into into the the phosphate phosphate buffer buffer (Figure (Figure2 ). 2).The The step step current current responses responses were were observed observed under under the the potential potential of of− −0.20.2V V (with (with a a response response timetime from from 85 85 to to 42 42 s), s), suggesting suggesting the the capability capability of of the the miniature miniature sensing sensing platform platform to to detect detect thethe tyrosine tyrosine residue-bearing residue-bearing peptidespeptides withoutwithout regenerating regenerating the the electrode electrode surface. surface. However, How- ever,following following the successive the successive addition addition of c-Src of c-Src substrate substrate 1, the 1, responses the responses decreased decreased gradually. grad- It ually.may beIt may due tobe thedue increase to the increase in detection in detection volume duringvolume the during successive the successive measurements measure- that mentslead tothat dilution lead to of dilution the peptide of the concentrations. peptide concentrations. This hypothesis This hypothesis could be could demonstrated be demon- by stratedplotting by the plotting responses the responses curves of c-Srccurves substrate of c-Src 1 substrate concentrations 1 concentrations vs. current responsesvs. current from re- sponsesthe results from of the Figure results2. Since of Figure the volume 2. Since of the electrolyte volume expandedof electrolyte from expanded 18 µL to from 32 µL 18 upon μL tothe 32 successive μL upon the injection successive of c-Src injection substrate of c-Src 1 stock substrate solution, 1 stock the ﬁnal solution, concentration the final of concen- peptide trationafter each of peptide addition after could each then addition be calculated could then by be multiplying calculated the by correspondingmultiplying the dilution corre- spondingfactor. As dilution shown factor. in Figure As shown S2, the in current Figure responsesS2, the current of c-Src responses substrate of c-Src 1 was substrate linearly 1proportional was linearly toproportional i-t concentration to i-t concentration after adjustment after withadjustment an R2 ofwith 0.998. an R The2 of 0.998. developed The developedminiature miniature tyrosinase-based tyrosinase-based tyrosine kinase tyrosine sensing kinase platform sensing exhibited platform a exhibited repeatability a re- of peatability6.6% RSD of (Relative 6.6% RSD standard (Relative deviation) standard fordeviation) a repetitive for a repetitive measurement measurement of 50 µM of c-Src 50 μsubstrateM c-Src substrate I (n = 6) (FigureI (n = 6) S4).(Figure S4).

FigureFigure 2. 2. TheThe current–time current–time curves curves of c-Src of c-Src substrate substrate 1 on 1 the on miniature the miniature sensing sensing platform. platform. Two mi- Two crolitermicroliter c-Src c-Src substrate substrate 1 stock 1 stock solution solution (500 (500 μM)µM) were were successively successively added added into into 100 100 mM mM phosphate phosphate buffer,buffer, pH pH 6.5. 6.5. The The initial initial volume volume of ofphosphate phosphate buffer buffer is 18 is 18μL.µ TheL. The i-t responses i-t responses of peptide of peptide were were monitored under a potential of −0.2 V vs. Ag/AgCl. monitored under a potential of −0.2 V vs. Ag/AgCl.

TheThe specificity speciﬁcity of of the the miniature miniature sensing sensing plat platformform to to recognize recognize peptide peptide with with tyrosine tyrosine residuesresidues was was further further demonstrated demonstrated by by alternately alternately adding adding 10 10 μµMM FLT3, FLT3, a a Her2 Her2 peptide peptide substrate,substrate, and and 100 100 μµMM kemptide, kemptide, and and a aPKA PKA peptide peptide subs substratetrate into into the the phosphate phosphate buffer buffer (Figure(Figure S3A, S3A, Supplemental Supplemental information). information). The The electrochemical electrochemical responses responses occurred occurred only only whenwhen FLT3 FLT3 was was added added into into the the phosphate phosphate buffer buffer.. Similar Similar results results were were also also observed observed when when ELMO-Y511,ELMO-Y511, a aHck Hck peptide peptide substrate, substrate, and and kemp kemptidetide were were alternately alternately added added (Figure (Figure S3B). S3B). Compared to the previously reported experimental setup [38], a smaller detection volume is needed for the developed miniature sensing platform. The electrolyte required for analysis was greatly reduced from 10 mL to 0.02 mL. Biosensors 2021, 11, x FOR PEER REVIEW 5 of 11

Biosensors 2021, 11, 240 Compared to the previously reported experimental setup [38], a smaller detection volume 5 of 11 is needed for the developed miniature sensing platform. The electrolyte required for analysis was greatly reduced from 10 mL to 0.02 mL. 3.2. Detection of Her2 and Hck Activity on the Miniature Sensing Platform 3.2. Detection of Her2 and Hck Activity on the Miniature Sensing Platform Previously, the miniature tyrosine kinase sensing platform was shown to be able Previously,to detect peptidethe miniature substrates tyrosine of protein kinase se tyrosinensing platform kinase, i.e.,was ELMO-Y511 shown to be andable FLT3.to To detect peptidefurther elucidatesubstrates theof protein capability tyrosine of miniature kinase, i.e. sensing ELMO-Y511 platform and in FLT3. detecting To further the tyrosine elucidatekinase the capability activity the of dose-dependentminiature sensing responses platform andin detecting time-dependent the tyrosine phosphorylation kinase ac- of tivity theELMO-Y511 dose-dependent and FLT3 responses were investigated. and time-dependent As shown phosphorylation in Figure3, ELMO-Y511 of ELMO-Y511 (closed circle) and FLT3and were FLT3 investigated. (open circle) As could shown be detectedin Figure in 3, a ELMO-Y511 linear range (closed of 10 to circle) 200 µ Mand with FLT3 the R2 of 2 (open circle)0.997 andcould 0.991, be detected respectively. in a linear range of 10 to 200 μM with the R of 0.997 and 0.991, respectively.

Figure 3.Figure Dose 3.responsesDose responses of ELMO-Y511 of ELMO-Y511 and FLT3 and peptides. FLT3 peptides. The electrochemical The electrochemical responses responses of of various various concentrationsconcentrations (0,(0, 10,10, 15, 30, 100 and 200 µμM) of ELMO-Y511 (closed circle) circle) and and FLT3 FLT3 (open circle) (open circle)were were determined determined on the on miniature the miniature sensing sensing platform. platform. The dataThe isdata mean is mean± S.D ± ofS.D three of three independent independentexperiments. experiments.

A time-dependentA time-dependent phosphorylation phosphorylation of ELM of ELMO-Y511O-Y511 and and FLT3 FLT3 peptides peptides by by Hck (10(10 U/mL) U/mL) andand Her2 (10 U/mL), respectively, respectively, was was also also determined determined at at 30 30 °C◦C for for 0, 0, 10, 10, 30, 30, 60, 60, 90, 90, and and 120120 min min (Figure (Figure 4).4 ).The The reductive reductive current current of of ELMO-Y511 ELMO-Y511 and and FLT3 FLT3 peptides peptides without without phos- phosphorylationphorylation were were 4.45 4.45 ×× 1010−7 −±7 6.70± 6.70 × 10×−9 10A −and9 A 4.48 and 4.48× 10−×7 ±10 3.03−7 ±× 103.03−9 A,× respectively.10−9 A, respectively. Upon theUpon phosphorylation the phosphorylation with tyrosine with tyrosine kinases, kinases, e.g. Hck e.g., and Hck Her2, and the Her2, reductive the reductive current current of ELMO-Y511of ELMO-Y511 and FLT3 and peptides FLT3 peptides (i.e., 3.27 (i.e., × 3.2710−7 ±× 2.1310− ×7 ±10−2.138 A and× 10 3.38−8 A × and10−7 3.38± 1.13× ×10 −7 ± − 10−8 A, 1.13respectively)× 10 8 A, reduced respectively) about 25% reduced after about 10 min 25% reaction after 10and min reduced reaction over and 50% reduced (i.e., over − − − − 1.57 × 1050%−7 ± (i.e.,2.75 1.57× 10×−8 A10 and7 ± 1.652.75 ×× 1010−7 ± 81.60A and × 10 1.65−8 A,× respectively)10 7 ± 1.60 ×after10 308 A, min respectively) reaction. after After 9030 min min reaction.phosphorylation, After 90 min the phosphorylation, electrochemical theresponses electrochemical of both responses peptides ofreached both peptides −8 −9 plateaureached with a reductive plateau with current a reductive of 2.78 current× 10−8 ±of 4.26 2.78 × ×10−109 for ±ELMO-Y5114.26 × 10 andfor 4.52 ELMO-Y511 × 10−8 and −8 −9 ± 3.06 ×4.52 10−9 ×for10 FLT3.± This3.06 ×result10 suggestsfor FLT3. that This the result activity suggests of Hck that and the Her2 activity can be of detected Hck and Her2 on the canminiature be detected sensing on theplatform miniature in a sensingkinase reaction platform for in as a kinase short as reaction 10 min. for as short as 10 min. The phosphorylation of ELMO-Y511 and FLT3 peptides by various activities (0, 1, 5, 10, 50, and 100 U/mL) of Hck and Her2, respectively, was carried out at 30 ◦C for 30 min prior to the electrochemical measurement. As shown in Figure5, the phosphorylation of peptides increased linearly with the logarithm of the activity of Hck (Figure5A and inset; R2 = 0.972) and Her2 (Figure5B and inset; R 2 = 0.941) with the lowest detection limit of 1 U/mL (S/N ≥ 3) to Hck and Her2. Biosensors 2021, 11, x FOR PEER REVIEW 6 of 11

Biosensors 2021, 11, 240 6 of 11 Biosensors 2021, 11, x FOR PEER REVIEW 6 of 11

Figure 4. Time-dependent phosphorylation of peptide substrates by Hck and Her2. The phosphorylation of 100 μM ELMO-Y511peptide (close circle) and 100 μM FLT3 peptide (open circle) were performed by Hck (10 U/mL) and Her2 (10 U/mL), respectively, at 30 °C for 0, 10, 30, 60, 90 and 120 min. At each time point, 2-μL of reaction mixture was withdrawn and subjected to electrochemical measurement under the potential of −0.2V. The data is mean ±S.D of three independent experiments.

FigureFigureThe 4. 4. Time-dependentTime-dependent phosphorylation phosphorylation phosphorylation of ELMO-Y511 of of peptide and peptide FLT3 substrates substrates peptides by by Hckby Hck various and and Her2. Her2. ac tivitiesThe The phosphor- phospho- (0, 1, 5, ylationrylation10, 50, of and of 100 100 100 μMµM U/mL)ELMO-Y511peptide ELMO-Y511peptide of Hck and (closeHer2, (close circle) respectively, circle) and and 100 100 μwasMµ MFLT3 carried FLT3 peptide peptide out (openat (open30 circle)°C circle)for were 30 were min performedperformedprior to the by by electrochemicalHck Hck (10 (10 U/mL) U/mL) and andmeasurement. Her2 Her2 (10 (10 U/mL), U/mL), As re shownspectively, respectively, in Figure at 30 at °C 30 5, ◦for Cthe for0, phosphorylation10, 0, 10,30, 60, 30, 90 60, and 90 and of 120120peptides min. min. At Atincreased each each time time linearlypoint, point, 2-μ 2-withLµ ofL reaction ofthe reaction logarithm mixture mixture of was the was with activity withdrawndrawn of and Hck andsubjected (Figure subjected to 5A electro- and to electro- inset; 2 2 chemicalchemicalR = 0.972) measurement measurement and Her2 under under(Figure the the 5B potential potential and inset; of of −−0.2V. R0.2 = V. The0.941) The data data with is is mean meanthe ±Slowest±.DS.D of three ofdetection three independent independent limit of 1 experiments. experiments.U/mL (S/N ≥ 3) to Hck and Her2. The phosphorylation of ELMO-Y511 and FLT3 peptides by various activities (0, 1, 5, 10, 50, and 100 U/mL) of Hck and Her2, respectively, was carried out at 30 °C for 30 min prior to the electrochemical measurement. As shown in Figure 5, the phosphorylation of peptides increased linearly with the logarithm of the activity of Hck (Figure 5A and inset; R2 = 0.972) and Her2 (Figure 5B and inset; R2 = 0.941) with the lowest detection limit of 1 U/mL (S/N ≥ 3) to Hck and Her2.

FigureFigure 5.5.Phosphorylation Phosphorylation of of ELMO-Y511 ELMO-Y511 and and FLT3 FLT3 peptides peptides by by various various activities activities of Hck of Hck and and Her2. Her2. The kinaseThe kinase reaction reaction was performedwas performed in kinase in kinase reaction reaction mixture mixture containing containing 0.5 mM 0.5 ATP,100mM ATP,100µM ELMO-Y511 μM ELMO-Y511 or FLT3 or peptideFLT3 peptide and various and various activities ac- (0,tivities 1, 5, 10,(0, 50,1, 5, 100 10, U/mL) 50, 100 ofU/mL) Hck (Aof) Hck and Her2(A) and (B). Her2 Reaction (B). Reaction was performed was performed at 30 ◦C forat 30 30 °C min. for Subsequently, 30 min. Subsequently, reaction mixturereaction(2 mixtureµL) was (2 subjected μL) was tosubjected electrochemical to electrochemical measurement measurement under the potentialunder the of potential−0.2 V. The of − inset0.2 V. of The each inset panel of is each the semi-logpanel is the plot semi-log of the same plot setof the of data. same The set dataof data. is mean The data± S.D is ofmean three ± S.D independent of three independent experiments. experiments.

3.3.3.3. The Effect ofof InhibitorsInhibitors toto thethe ActivityActivity ofof HckHck andand Her2Her2 Figure 5. Phosphorylation of ELMO-Y511ProteinProtein tyrosinetyrosineand FLT3 kinasekinase peptides inhibitorsinhibitors by various areare activities widelywidely of usedused Hck in inand thethe Her2. laboratorylaboratory The kinase toto elucidate elucidatereaction thethe was performed in kinase reactionsignalingsignaling mixture pathwaypathway containing asas wellwell 0.5 mM asas inin ATP,100 thethe clinicclinic μM totoELMO-Y511 treattreat cancers.cancers. or FLT3 TheThe peptide effecteffect ofofand inhibitorsinhibitors various ac- cancan bebe tivities (0, 1, 5, 10, 50, 100 U/mL)accessedaccessed of Hck byby thethe(A) decreaseanddecrease Her2 inin(B ). thethe Reaction proteinprotein was tyrosinetyrosine performed kinasekinase at 30 activityactivity °C for after after30 min. treatment.treatment. Subsequently, Therefore, reaction mixture (2 μL) wasthethe subjected capabilitycapability to electrochemical of of the the developed developed measurement miniature miniature under tyrosine tyro thesine kinasepotential kinase sensing of sensing −0.2 platformV. platformThe inset in studying ofin eachstudying the panel is the semi-log plot of inhibitorythe sameinhibitory set effect of data.effect of Src-I1,Th ofe dataSrc-I1, the is mean inhibitor the inhibitor ± S.D of of Src three of family Src independent family kinases kinases experiments. [40], and[40], CP724714, and CP724714, the Her2 the speciﬁcHer2 specific inhibitor inhibitor [41], on[41], the on corresponding the correspondi kinasesng kinases was was studied. studied. As As shown shown in Figurein Figure6, 3.3.the The activity Effect of of Hck, Inhibitors a Src to family the Activity kinase, of couldHck and be Her2 suppressed about 88% in the presence of 176Protein nM Src tyrosine I-1, whereas kinase onlyinhibitors about are 13% widely of Her2 used activity in the waslaboratory inhibited. to elucidate In contrast, the signaling20 nM CP-72471 pathway could as well inhibit as in Her2 the clinic activity to treat by about cancers. 91%, The but effect only slightlyof inhibitors affected can thebe accessedHck activity by the by decrease around 12%. in the protein tyrosine kinase activity after treatment. Therefore, the capability of the developed miniature tyrosine kinase sensing platform in studying the inhibitory effect of Src-I1, the inhibitor of Src family kinases [40], and CP724714, the Her2 specific inhibitor [41], on the corresponding kinases was studied. As shown in Figure Biosensors 2021, 11, x FOR PEER REVIEW 7 of 11

6, the activity of Hck, a Src family kinase, could be suppressed about 88% in the presence of 176 nM Src I-1, whereas only about 13% of Her2 activity was inhibited. In contrast, 20 Biosensors 2021, 11, 240 nM CP-72471 could inhibit Her2 activity by about 91%, but only slightly affected the7 Hck of 11 activity by around 12%.

FigureFigure 6. 6. EffectEffect of of inhibitors inhibitors on on the the activity activity of of Hck Hck and and Her2 Her2 protein protein kinase kinases.s. The The phosphorylation phosphorylation reactionsreactions were were performed performed in in a a kina kinasese reaction reaction mixture mixture containing containing 10 10 U/mL U/mL of ofHck Hck (closed (closed bar) bar) or or Her2Her2 (open (open bar) bar) and and 100 100 μMµM ELMO-Y511 ELMO-Y511 (for (for Hck) Hck) or FLT3 or FLT3 (for (for Her2) Her2) with with or without or without 176 nM 176 Src- nM I1Src-I1 or 20 or nM 20 CP-724714. nM CP-724714. After After reaction, reaction, 2 μL 2reactionµL reaction mixture mixture were were subjected subjected to electrochemical to electrochemical measurementmeasurement on on the sensing platfo platform.rm. The relative responses uponupon phosphorylationphosphorylation ininthe thepresence pres- enceand absenceand absence of inhibitors of inhibitors were were calculated calculated and an presentedd presented as theas the mean mean± S.D ± S.D of of three three independent independent experiments. (* denote significantly different from the activity of “kinase only” at p < 0.05). experiments. (* denote signiﬁcantly different from the activity of “kinase only” at p < 0.05).

3.4.3.4. Interference Interference Effect Effect of of Cultural Cultural Medium Medium to to the the Protein Protein Tyrosine Tyrosine Kinase Activity Generally,Generally, most of thethe proteinprotein tyrosinetyrosine kinases kinases are are either either membrane membrane bound bound or or residing resid- ingin thein the cytoplasm. cytoplasm. Hence, Hence, it it is is usually usually required required to to prepare prepare samples samples fromfrom cellularcellular extract extract forfor protein protein kinase kinase assay. assay. To To understand understand the the effe effectct of the of thecell remnants cell remnants or intracellular or intracellular con- stituentsconstituents in causing in causing the theinterference interference with with the the electrochemical electrochemical measurement, measurement, the the culture culture mediummedium (DMEM (DMEM containing containing 10% 10% fetal fetal bovine bovine serum) serum) was was used used to prepare to prepare the thekinase kinase re- actionreaction mixture mixture that that contains contains peptide peptide substrate, substrate, i.e., i.e.,ELMO-Y511 ELMO-Y511 or FLT3, or FLT3, and and protein protein tyrosinetyrosine kinases, kinases, i.e., i.e., Hck Hck or or Her2. Her2. The The redu reductivective current current of of ELMO-Y511 ELMO-Y511 and and FLT3 FLT3 alone alone − − − − waswas 4.15 4.15 ×× 1010−7 ± 72.66± 2.66 × 10−×8 A10 (closed8 A (closed bar) and bar) 4.57 and × 10 4.57−7 ± ×3.2510 × 107 ±−8 A3.25 (open× 10 bar),8 A respec- (open tivelybar), respectively(Figure 7, peptide (Figure only).7, peptide When mixed only). with When the mixed culture with medium, the culture the electrochemical medium, the responseselectrochemical of ELMO-Y511 responses and of ELMO-Y511 FLT3 (peptide and + FLT3culture (peptide medium) + culture decreased medium) slightly decreased to 3.87 −7 −8 −7 −8 ×slightly 10−7 ± 4.34 to 3.87× 10−×8 A10 and ±4.074.34 × 10×−710 ± 3.78A × and 10−84.07 A, respectively.× 10 ± 3.78 This× result10 A,suggests respectively. that a cellThis culture result suggestsmedium thator even a cell a cell culture crude medium extract ormay even not a affect cell crude the electrochemical extract may not meas- affect urementthe electrochemical of peptides measurementon the sensing of platform. peptides on the sensing platform. In contrast, the activity of Hck and Her2 was moderately affected by the culture medium. This is demonstrated by the ﬁnding that the phosphorylated form of ELMO-Y511 (dark bar) and FLT3 (bright bar) decreased in the kinase reaction mixture containing the culture medium (Figure7). The ELMO-Y511 and FLT3 peptides phosphorylating without the culture medium exhibited a response (peptide + kinase) of 8.26 × 10−8 ± 5.31 × 10−9 A and 8.51 × 10−8 ± 8.39 × 10−9 A, respectively; while in the presence of culture medium, the responses of peptides (kinase + culture medium) changed to 1.63 × 10−7 ± 2.44 × 10−8 A and 1.97 × 10−7 ± 2.88 × 10−8 A, respectively. This result indicates that the kinase activity of Hck and Her2 was suppressed 33% and 43%, respectively, by the culture medium. Although the exact mechanism underlying the medium-mediated inhibition of kinase activity is not clear, the presence of kinase inhibitors, phosphatases, proteases and/or thiol compounds, in the culture medium is postulated. Phosphotyrosine phosphatases are known to remove the phosphate group from the phosphotyrosines; while proteases contaminants can degrade protein kinases. The contamination of both substances may result in the underestimation of kinase activity. Thiol compounds, such as cysteine and glutathione, could block the reaction of tyrosinase by forming the inactive conjugates from the intermediates [42]. To avoid the inﬂuence of cellular components on the kinase reaction, the reaction mixture for protein tyrosine kinase reactions is suggested to be reformulated, such as adding phosphatase inhibitors and sample pretreatment. BiosensorsBiosensors 20212021,, 1111,, 240x FOR PEER REVIEW 88 of of 11

Figure 7. InterferenceInterference effect effect of of culture culture medium medium on on the the detection detection of peptides of peptides of protein of protein tyrosine tyrosine kinases. Peptide stock solution and and protein protein kinase stock stock solution solution were were diluted diluted with with the the cell cell culture culture medium in aa ratioratio ofof 1:1.1:1. TheThe phosphorylationphosphorylation was was performed performed in in a kinasea kinase reaction reaction mixture mixture containing con- 10taining U/mL 10 ofU/mL Hck of (closed Hck (closed bar) or bar) Her2 or (openHer2 (open bar)and bar) 100 andµ 100M ELMO-Y511 μM ELMO-Y511 (for Hck)(for Hck) or FLT3 or FLT3 (for (forHer2). Her2). The The data data is mean is mean± S.D ± S.D of three of three independent independent experiments. experiments. (* p (*< p 0.05). < 0.05).

4. ConclusionsIn contrast, the activity of Hck and Her2 was moderately affected by the culture medium.In This this is work, demonstrated a simple andby the label-free finding miniaturethat the phosphorylated sensing platform form for of detecting ELMO-Y511 the (darkactivity bar) of and protein FLT3 tyrosine (brightkinases bar) decreased was developed in the kinase and characterized. reaction mixture Compared containing to thethe previousculture medium experimental (Figure setup 7). The [38 ELMO-Y511] and published and FLT3 reports peptides (Table 1phosphorylating), the newly developed without platformthe culture is simplemedium in exhibited design, easy a respon to operate,se (peptide and requires + kinase) only of 1–28.26µ L× 10 samples−8 ± 5.31 for × kinase10−9 A andactivity 8.51 assay.× 10−8 ±With 8.39 × the 10− reduction9 A, respectively; of detection while volume,in the presence the sample of culture volume medium, required the responsesfor analysis of waspeptides also signiﬁcantly(kinase + culture decreased medium) from changed 20 µL to to 1.63 2 µL. × 10 Simple−7 ± 2.44 in × design 10−8 A andand 1.97easy × operation 10−7 ± 2.88 are × 10 also−8 A, the respectively. advantages This of the result current indica experimentaltes that the kinase setup. activity Thesestudies of Hck andshowed Her2 that was the suppressed current experimental33% and 43%, setup respec exhibitstively, by great the potentialculture medium. for the Although detection theof biological exact mechanism samples underlying that are usually the medium-media rare and expensiveted inhibition to acquire of kinase in large activity quantity. is not Theclear, unique the presence tyrosinase-based of kinase inhibitors, detection ph mechanismosphatases, [38 proteases] allows theand/or developed thiol compounds, miniature insensing the culture platform medium to detect is postulated. different proteinPhosph tyrosineotyrosine kinases phosphatases on the sameare known electrodes. to remove This theis demonstrated phosphate group by the from observation the phosphotyrosin that both Hckes; while and Her2 proteases activity contaminants can be successively can de- gradedetected protein without kinases. changing The contamination electrodes. The of platformboth substances was also may effective result in in the assessing underesti- the mationspeciﬁcity of kinase of inhibitors activity. on Thiol different compounds, tyrosine su kinases,ch as cysteine indicating and thatglutathione, the platform could can block be theused reaction to monitor of tyrosinase and screen by the forming effect ofthe drugs inactive on different conjugates tyrosine from kinasesthe intermediates in a short time.[42]. ToIn summary,avoid the influence the developed of cellular tyrosine components kinase sensing on the platformkinase reaction, exhibits the a great reaction potential mixture to forbe aprotein powerful tyrosine tool forkinase the reactions detection is of suggest proteined tyrosine to be reformulated, kinase activity, such the as adding screening phos- of phatasetyrosine inhibitors kinase-based and drugs, sample and pretreatment. clinical diagnoses.

Table 1.4. ComparisonConclusions of the performance of various tyrosine kinase biosensors.

In this work, a simple andSample label-free miniature sensing platform for detecting the Protein Linear Limitation activity of proteinReusability tyrosine kinasesVolume was developed and characterized. Compared to the Electrode Types Working Mechanism Tyrosine Range of of Ref. (R.S.D.) Required previous experimental setup [38] and publisKinaseshed reportsDetection (Table 1), theDetection newly developed platform is simple in design, easy( µtoL) operate, and requires only 1–2 μL samples for kinase activity assay. With the reduction of detectionSrc volume, the N.D. sample volume N.D. required for Miniature Tyrosinase-based Tyr High This 1–2 µL Hck 1–100 U/mL 1 U/mL Tyrosinase/CFP Oxidationanalysis was also significantly(6.6%) decreased from 20 μL to 2 μL. Simple in design andstudy easy operation are also the advantages of the curreHerBnt experimental 1–100 U/mL setup. These 1 U/mL studies showed Peptide- that the current experimentalLow setup exhibits great potential for the detection of biological immobilized AuNP-based redox response 25 µL Src N.A. 5 U/mL [32] samples that are usually(N.A.) rare and expensive to acquire in large quantity. The unique tyro- SPCE sinase-based detection mechanism [38] allows the developed miniature sensing platform to detect different protein tyrosine kinases on the same electrodes. This is demonstrated Biosensors 2021, 11, 240 9 of 11

Table 1. Cont.

Sample Protein Linear Limitation Reusability Volume Electrode Types Working Mechanism Tyrosine Range of of Ref. (R.S.D.) Required Kinases Detection Detection (µL) MWCNT High Direct oxidation of Tyr 20 µL Src N.A. 5 U/mL [35] -modiﬁed SPCE (N.A.) Graphene- Graphene-assisted direct High 0.26 to modiﬁed glassy 20 µL Src 0.087 nM [37] oxidation of Tyr (N.A.) 33.79 nM carbon electrode Tyrosinase-based Tyr High 1.9–237.6 Tyrosinase/CFP 20 µL Src 0.23 U/mL [38] Oxidation (2.87%) U/mL 4-mercaptophenylboronic Peptide- acid (MPBA)MPBA-assisted Low immobilized – Src 10–80 ng/mL 1.2 ng/mL [43] AgNP aggregates-based (N.A.) Gold electrode redox response Peptide- Os(bpy) +2-mediate Tyr Low immobilized ITO 3 – EGFR N.A. 1 U/mL [44] oxidation (N.A.) electrode N.D., Not determined; N.A., Not applicable; MPBA, 4-mercaptophenylboronic acid; SPCE, screen-printed carbon electrode.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/bios11070240/s1, Figure S1: The structure of the miniature protein tyrosine kinase sensing platform., Figure S2: The response curve of c-Src substrate 1 concentration vs. current responses; Figure S3. The chronoamperometric measurement of substrate peptides for Hck and Her2; Figure S4. Reproducibility of tyrosine kinase sensing platform. Author Contributions: Conceptualization, C.-J.Y.; methodology, L.-Y.W.; validation, W.L., C.-J.Y. and B.-F.L.; formal analysis, L.-Y.W., W.L.; investigation, L.-Y.W.; resources, C.-J.Y., C.-C.C., B.-F.L.; data curation, C.-J.Y.; writing—original draft preparation, L.-Y.W., C.-J.Y.; writing—review and editing, C.-J.Y., B.-F.L., L.-V.K.; visualization, W.L.; supervision, C.-J.Y.; project administration, C.-J.Y.; funding acquisition, C.-J.Y., C.-C.C., B.-F.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the grants MOST 107-2314-B-009-002, MOST 109-2314-B- 009-002, MOST 109-2327-B-010-005 and MOST 109-2927-I-009-003 from the Ministry of Science and Technology (MOST), Taiwan and the University of Malaya Research Grant-SATU (ST003-2020). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work was financially supported by the Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) from The Featured Areas Research Center Program within the frame- work of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and the MOST, Taiwan. Conflicts of Interest: The authors declare no conflict of interest.

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