Scribd Logo
Upload

Welcome to Scribd!

  • Evaluation of Enzyme Immobilization Methods For Paper-Based Devices-A Glucose Oxidase Study - Elsevier Enhanced Reader

    Uploaded by

    onur ateş
    11 views9 pages

    Original Title

    Evaluation of enzyme immobilization methods for paper-based devices—A glucose oxidase study _ Elsevier Enhanced Reader

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    11 views9 pages

    Evaluation of Enzyme Immobilization Methods For Paper-Based Devices-A Glucose Oxidase Study - Elsevier Enhanced Reader

    Uploaded by

    onur ateş

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    of 9
    Journal of Pharmaceutical and Biomedical Analysis 17 (2016)551-59 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/|pbs Evaluation of enzyme immobilization methods for paper-based devices—A glucose oxidase study Emilia Witkowska Nery®:**, Lauro T. Kubota®** @-= + Deparment of Analytical Chemistry, ste of Chemisty UNICAMP, RO. Box 6184 13084971 Campinas SP, rat ® national sate of Scene and Technolgy oanays, Instat of Cemsry-UNICANE, PO. Bax 6154, Campinas, Br ARTICLE INFO ABSTRACT ‘rice ion Received 73 Jly 2015, Received in revise form 26 August 2015 [Aecepted 28 August 2015; ‘Rie online 1 October 2015 entrapment in get Keywords Tab-on paper apr-based analytical device Bioactive paper Enzyme immobilization aoe 1. Introduction Paper-based sensors accompany analytical chemistry for cen- turies in form of spot-tests, litmus paper and lateral flow assays based on nitrocellulose but just in the last few years this hum ble substrate was rediscovered as 2 platform for integrated microfluidics. Growing popularity of paper as a substrate for sensors can be attributed to its unique mechanical (flexibility, lightness, soft texture) and structural properties (absorbancy, air permeability, high surface-to-volume ratio, capillary action) as well as its natural origin (biocompatibility. ease of steriiza~ tion, chirality, biodegradability), In the last few years numerous microfluidic paper-based analytical devices based on optical (colorimettic, fluorimetric, SERS surface-enhianced Raman spec- ‘troscopy, chemiluminescenece, transmittance) and electrochemical detection methods (voltammetric, potentiometric, conductivity measurements) were presented in the literature, Detailed reviews ‘of recent advances, methods of fabrication as well as the history of paper-based analytics are already available 1-2), Covtespoading author at: Department of Analytical Chemisty, Insitute of ‘Chemistry UNICAMP, PO Box 6154, 13084971 Campinas, Bal * Coresponding author at: Department of Analytical Chemisy, taste ‘of Chemisty-UNICAMP, PO, ox 6134, TaOR4G71 ‘Campinas, SP, Bsa Fan 55 199521 3127, mall address ela pery@igm nicamp.h (EW. Nery, Jborasign uniearnp.br(LT Kubota) px ot on 10.1016), p40 2015 08.01 (0731-7095j0 2015 Esevir RV All ight eserved, Paper: based sensors gained i tems, often destined to resource limited settings is based on enzymatic reactians. Choe ofan adequate immobilization method could significantly proiong the shel-life of such sensors, especially in applica: tions. where exposure to high temperatures during storage and transport is more than a threat, We are Seeking fo compare a variety of immobilization methods based on different phenomena (adsorption, eroeneapsulation,covae ‘dase was used 35 4 model enzyme. Enzymatic activity of immobilized samples was accompanied for 3 Deriod of 24 weeks considering two sets of samples, one stored in 4 "Cand other in ambient temperature. few years Alarge number of sy3- a explosive attention during th linkage) with coal of 33 methods tested. Clucose o "© 2015 Elsevier BV. Al rights reserved Researchers often emphasize how properties of paper make it an jdeal substrate for sensors for the developing world, Nev= ertheless it as to be remembered that those resource limited settings often lack proper refrigeration during storage and trans- port, can suffer from energy loss. lack of supplies ete. Even with, so many reports describing new paper-based platforms being pub- lished, few of them explore their storage stability. A large number of paper-based sensors are based, at least to some extent on enzy~ ‘matic reactions and protein immobilization methods are known to increase shelf life of such sensors, In fact properly designed immobilization can be beneficial to almost all enzyme properties, such as activity, specificity, selectivity, reduction of inhibition and aforementioned stability. Stability is dictated by the nature and ‘number of bonds between the enzyme and the support, degree of confinement, immobilization conditions and microenvironment created. Immobilized enzymes can gain new interesting properties, as higher volumetric activity or optimized performance under spe- cifle conditions (eg, organic solvents, alkaline, acidic conditions elevated temperatures). Other example is the possible reusability. enzyme can be recycled and used again reducing the overall cost. Enzyme attached to a support can form selective sorbents for pro- tein purification or can be used in systems for controlled release of drugs. On the other hand, poorly designed, immobilization can in some cases lead to decreased stability. Because of this we would like to compare the impact fon storage stability of a variety of immobilization methods based on different phenomena from physical adsorption, through ss EW. Nery, LT Rubra /Joural of Pharmacare and medical Atay 117 (2016) 51-559 ‘entrapment in gel, encapsulation to covalent linkage (33 meth- ‘ods tested), Study also includes use of blockers (glycine, bovine serum albumin [BSA}), surface treatment (adsorption on a layer of polymer), use of protectants (mannitol and trehalose). Nowadays, ‘genetically modified enzymes incorporating appropriate binding ‘domains, which adhere spontaneously to cellulase and/or he: cellulose can be used for biochemical coupling with the suppor. In case of paper many engineered enzymes with cellulose bind- ing domains are already commercially available, but their price is usually higher. Because of lesser availability of such enzymes, immobilization methods based on cellulose binding domains were not included in cis study. Glucose oxidase (GOx) was used as a model enzyme. GOx is, ‘multimetric and as other enzymes from oxidoreductase family was shown to dissociate under hydrostatic and osmotic pressure, increased temperature and extreme pH. However stabilization ‘could be accomplished by means of immobilization [4]. We chose ‘glucose oxidase because assessing glucose levelsis among the most important analytical tasks, with glucose tests forming about 40% of all blood tests in 2011 [5] and approximately 85% ofthe world mar- ket of biosensors in 2005 6}. Glucose oxidase is frequently selected as a model enzyme to investigate new immobilization systems [7 During this study the amount of protein immobilized was quan- tified by diffuse reflectance spectroscopy (200-280nm) and the ‘enzymatic activity was accessed by the change in absorbance at 570 1nm (potassium iodide-starch method for hydrogen peroxiee) ‘We think that our experimental review willbe a valuable addition to several recently published literature-based reviews concerning, immobilization on cellulose [8,9 2, Materials and methods Immobilization procedures are resumed in Table 1. Methods, are divided in 5 groups: physical adsorption, entrapment in gel layer-by-layer, covalent binding and encapsulation. Glucose oxi- dase from Aspergillus niger 18.5U)mg (Sigma-Aldrich) was used for all experiments. 2.1. Enzymatic activity, optimization of essay conditions First, pH was optimized for the glucose oxidize reaction with, starch-iodide reagent. Tests were performed in solution, using Ultraspee 2000 UVivis spectrophotometer. Starch-iadide reagent ‘was prepared by mixing KI 3 mM, NagMoO, 2.5 mM and starch 1OmgimL, which were dissolved by gentle heating. For the pH ‘optimization assay 0.8 mL of stareh-iodide reagent of appropriate pH, 1.0mL of PBS 0.2mM buffer, 0.2mL of SmM glucose solution ‘were mixed and quantification was performed after 10min from addition of the enzyme. Three samples were prepared for each PH tested, Subsequently a calibration curve for enzymatic assay in Solution was prepared (enzyme concentration 0.1-10 mg/mL) atpH 5.5. Enzyme concentration of 5 mg/ml was chosen for subsequent experiments on paper. 22. Enzyme immobilization methods ach sample was a square piece of Whatman no 1 paper with a 1 Tem samplearea defined with wax (XEROX Phaser printer)_ 10]. ‘Samples for each immobilization method were coded and mea- sured in random order. A 10 iL of solution was needed to wet the paper completely, if not stated otherwise this volume was used for all modifications. All solutions if not stated differently were pre- pared in PBS pH 5.5. In case of layer-by-layer methods paper was covered with 3 bilayers starting with anionic polymer. Inthe second and thid bilayer cationic polymer was mixed with Glucose oxidase to obtain final concentration of 25 mgm (92.5 U/mL} of enzyme. In the case of subsequent methods: EDC, EDC/NHS, entrapment in ammonium alginate and all encapsulation methods several batches ‘with different concentration of reagents and reaction times were prepared, Table | fists only the optimized conditions of aforemen- tioned methods. Incase ofencapsulation methods, smaller capsules ‘were dispersed on sample papers with a pipette, larger capsules ‘were transported manually on paper and left to dy. 23. Quantification of enzyme present inthe sample after immobilization In case of some immobilization methods (encapsulation and metivods that require washing steps) the final concentration of enzyme in the sample is not known. Therefore in order to com= pare activity of those samples it is necessary to quantify the Amount of protein present. Aromatic rings of amino acids cause an absorbance peak at 2801nm and peptide bonds are responsible for peak around 200 nm. Secondary, tertiary and quaternary structure, and therefore factors such as pH, ionic strength affect absorbance, A spectrophotometer, with varian integrating sphere designed for solid samples was used (Agilent Cary 500) with teflon serving as blank. For tests in solution calibration curves (0,3-20mgi/mL) were prepared for both Glucose oxidase and BSA (serving as a model pro- tein), three replicates were made for each concentration. For teston, paper, |x 1 cm devices descried earlier were modified with 10 pL ‘of sample, Each time before assessment of enzymatic activity paper devices were analyzed forthe amount of protein present. For tests on paper intensity around 280nm is too low. but pro- teins can be quantified by the absorption peak at 200nm (Fig. 1) Calibration curve was constructed for GOx on paper using inclina- tion of the spectrum in the region of 204-220 nm (N=3).Data show linear dependence for concentrations between 1 and 20mgimL GOx y=~0,0021 x +0.0004, R? 0.9999, 2.4. Impact of enzyme immobilization on stability during storage In all cases 3 samples with enzyme and 1 control without Glu- cose oxidase were prepared for storage in ambient temperature and, the same set for storage in 4°C foreach day of measurement. Sam- ples were measured after 1,3 days and 1,2,3,4,5,6,7,8, 10,12, 14, 16,20, 24 weeks of storage. In case of complete loss of activity tests ‘were terminated prematurely. Potassium iodide-starch method ‘as used for the evaluation of enzymatic activity, Each time I mL ‘of PBS buffer and 0.8 mL of potassium iodide-starch reagent were placed in the cuvette, after that the enayme madified paper was submerged in a way as to not to obstruct the light path, and 0.2 mL of glucose solution 5 mM was added. After 15 min the solution was mixed and the spectrum was taken. 25, Michaelis-Menten kinetics To analyze the metabolic activity of selected immobilization ‘methods Lineweaver-Burk curve described by Michaelis-Menten, kinetics was constructed, For this experiment only most promis ing immobilization methods were selected. Therefore tests were performed only with subsequent methods: enzyme in solution, adsorption, BSA blocking, entrapment in PVA, entrapment in starch, {DL sodium alginate-chitosan, LDL Hyaluronic acid—PEI, covalent linkage EDC/NHS. Enzymatic activity was evaluated accordingly to previous tests (1 mL PBS; 0.8mL of potassium iodide-starcl (02 ml glucose) for glucose concentrations equal 0, 0.5, 1,5. 10,50, 100, 200M. The 3 samples were measured for each concentra tion, Measurements were conducted with potassium iodide-starch, reagent and different concentrations of hydrogen peroxide (0.02: 0.05; 0.1 mM) to evaluate the extinction coefficient, EW. Rey, LT Kubota Jounal Pharmacericl and omdial nabs 17 (2006) 551-559 ss Table Inmobiization methods reviewed during this stu, with heir preparation procedure No Group eto Preparation procedre erence T ysl adsorpion Simple adsorpion TL ef GO. smgimt 25ujmt) - 2 [sAblocking WhLef CO. Smgiml afte dry 1pL of BSAS mg im 3 (Ona ayer afcolagen Whkofconfctonay gelatin OmgimL after dry 1OyLot 12), 0 5mgin 4 (Ona ayer of tenicacd yLestearicacd 475.mgil ater dey Lot ray cos Ssmgia 5 (na layer of polyvinyl TOyLofPVA 1LSmglol aferdry WuLdeCOx«Smgimk 4-16] coho! 6 ‘itnmannit!| 10uLof GO. 5mglmL- mannitol mgm wy 7 With halos WhLof CO Small ehalose 275 mg/m his} 5 Entrapment in go Instarch 04 ofCO-<5mgim starch 10mm ho} 5 Inchitosan 04 eF GO Smgimb - citasan 15 mgm: chitosan hist solution was fst prepared water with acetic acd ad [acer ditted with PBS and NasHPO, to obtain the desired a 0 mnpva MpLetCOxsmgimt- PVA Seglo ers] " Indextran WhLof CO «Smal dextran 2a fo} 2 Inammoniom algae JojLofCo™ Sng ammonium alginate Fm. Faia B inoue TO iLof Go Sma CMCT mein fao23] 1 ayer bylyer Siu algaate—chtosan Sdium algae aman chicosan Zao peal 8 odie Sodium agaae Amal, pollsine 2a, [aa25) lgiate—polyysine Sedum algnare—Pea Sodium lgaate Aman, PL 10mg 25271 Tyler aexd—chitosan Hyaluronic acid 2g, etosan 2g [asa] aluronic atd—PEL yaluronic acid 2mgjmi Pl oman (25.25) Palyiay Poly(einy slate) 2 mgm chitosan 2 mgm [2729] sulfate}ehiosan 2» olyacie acid}chitosin Poy acylic ae) 5.6 mg, chitosan 2m (27.30) 21 covaleneinkage ltarldehyde TOL of CO.» Smglmk, when dry 1OuLof glutaraldehyde rai} ‘cossnking 25% 2 ‘luraraldehyde {yi oF GO «Smgimk, when dry 10pL of sloaraldehyde Mosied method 21 cossinking with pycine 25% ater 1-30 samples sprayed with 1M lyin. ‘Blocking a Sehiaee Samples of paper were submerged in 100i KlOgfor 12h, (32) washed with water and ried witha paper rowel 10 pL {601mg was spotted two tines on each sample ‘within aninteralot Th After Th TORL of Gmg/mot [aijen was spate on each device which were ter hwashed several mes with water, 2" DC with line blocking WuLofEDC2O%mghml, CO » 1OmglmL wasspoted on co cach spl and etto rea for Thin dC Sabet ‘mote TOL EDC/GOX mixture was sped ade for 2h ‘n4°c. Alter that time TOpL of gyine was sported on each sample Fo rncinns TO Lot 150!mg/mt EDC, 28 maim NHS was sported and bal lotto eset for hia € After tha tine move 10 Lae sted and let to eact for 3h son 4 Subsequently Wutei CO. 10mgimL was spoted andalter2h more outcox. 26 Porassium periodate, Paper substrate immersed in hot 01 MID, for 130. im sthlenedamine, od ‘Washed with water to remove excess periodate and sluaralehie activation ‘mmersed ina solution of 125 mn of ethylenediamine fe 2:308 and in 2% oton of taal fo 1:20, ‘washed times with eel eter IDL of {60.10 mga was deposited on each sample 27 Encapsulation Sodium alginate Solution of 30mglmLofsodlum alginte aad 20 mg/mL of wast {Gox was ade dropwise to 555i of cacy under constant sig. 8 Sodium algnate-CMC Soluden of ISmglmtofsodiumalginet, 30mgimLofeMe (35) i 20mgin of COx was add drops 0 S54iL of {oti under constant stinine » Sodium lgnate—citosan Soltion of Smal of sam alate, 20mqimLof ru chitosan and 20mgimLof COx was ade dropwise to S5alLofcocl under constant string 20 re SmLofa solution of 120ml 4 wth Sgn, GOx bps} Wwasadded 0 25mLofyelohexane with Oat Triton 100 ander constant string, Sminater the emulsion was formed 12mg of sebacoyl chloe in 1.2m ffejlonenane was added othe matte Aer 10m of {eaction anther Sa of sebacolclovide TglaL was ‘ded and lef react for 10min, Reaction was topped ‘with 25 mf cyclohexane and the caples mashed with ss EW. Ney, LT. Kubota Joural of Pharmac and medical Analyt 117 (2016) 51-59 Table (Coniued) No Group Method Preparation procedure Reerence 3 “covalent azashmeni vo PEI Asthe capsules rom method 30d ot show any Modes method 0] capsules ‘azymatc activity they weve used a3 base covalently drach GOx by means of lutaalcehye ceslnking. For this purpose 10mL ofa suspension of PEL capsules rom the previlusstucy was mised with 1m. COD 10 mal 2nd dn fglttaldehide 25% and lett seat or Th Under constant string Capsules were washed with ‘stilled water: 2 Covalent attachment to "Sgofchromatgraphyslcabeadswas mined wethSmL (15) sien bead ‘of lOmalmi GOx and glraraletyse 25% ane 0 ect for Th Aterwards sed with water 2 Chitosan 1m fll chitosan, 20 mei Gl mistare ws bs} ade t 507 of cooking el with 05% of Tween Surfactant and emote After Smin SmLof _ltaraldehye 25% was added and et to eat for 15in {Oo mL fds water was wed to stop thereat, 3. Results and discussion 3.1. Impact of enzyme immobilization on stabitty during storage Figs. 2-6 resume the initial (24) activity of immobilization, samples (N=3) with normalized absorbance. Range of the results for the initial enzymatic activity was very large, two methods (LbL. alginate-chitosan and EDCNHS) presented absorbance higher than, the working range ofthe spectrophotometer (»3)in those two cases results presented were obtained after 48 hof storage ‘Table 2 restimes the impact of storage temperature and time ‘on enzymatic activity, results are shown as percentage of the initial activity observed for each method after 24h. Methods in ‘which activity dropped to O% after just 1 day of storage were rot included in the summary. Choice of enzyme immobilization ‘method will depend mostly on the application, and the character- istic in question: ong term stability stability in room temperature, ‘work in flow conditions, ease of preparation etc, Selected method should meet both catalytic (selectivity, stability, and productiv- ity) and non-catalytic (ease of separation, minimum leakage etc.) requirements. There is no general universally applicable method for enzyme immobilization, and also in this study it is dificult to cite one outstanding result For storage in 4°C for periods up to 8 weeks simple adsorption, in paper would probably be the choice. It shows rather low ini- tial activity (~17%) as compared with LDL sodium alginate-chitosan (luighest initial activity ofall the methods), but is extremely easy {o prepare and does not fequire any other chemicals except the enzyme, All adsorption methods showed rather low initial activ ity (from 7 to 30% as compared with LbL sodium alginate-chitosan ‘method BSA blocking method stands out, ait showed the longest storage stability regardless the temperature. itis easy to prepare and in resource Wimited settings powdered milk could be used instead of purified albumin, In case of entrapment methods very distinct results were ‘observed for simple methods where enzyme was mixed with the polymer and LbL. For storage in lower temperatures entrapment in dextran would be the recommended method, itis simple, inex pensive and showed activity above 75% ofthe initial value after 14 ‘weeks (4°C)—best results from all the methods tested. ‘Storage stability in room temperature Was highest for entrap- ‘ment in starch and chitosan (simple entrapment). Initial activity ‘was even lower for the simple entrapment methods than for adsorption, probably because of the lesser accessibility of the enzyme when enclosed in polymer. Simple entrapment meth- ods are extremely easy to prepare, on the contrary to LbL ‘which requires multiple addition and drying steps. Layer-by-layer ‘Wavelengthiom] Fig. 1. Quantifation ofthe amount of protein on paper—overad absorbance spectra, EW. Nery LT Kubora/ Jou hormaceurcal ond Moredial nabs 17 (2018) 951-559, 85 Table? Impact of time and temperature of storage on enzymatic activity (For interpretation of he reerences to coor inthis table legend he reader is refered the web version ofthis aticie) site Pam son Sa 4 3] ecg Rr Rr Rr lee ise RP Rr Rr a 386 EW. Nery, LT: Rubra /Joural of Pharmacare and medical Atay 1172016) 51-559 1 09 08 7 in he soni @skvcing Coben Seneadd PVA Nomi Tease too ‘8Reomtenperatre Fig. 2 Ina enzymatic acy of adsorption methods 1 a9 os °7 06 05 os 03 02 1 PAentapmert Stach Chitosan Alginate ate ‘|Roemtenpeatre Absorbance Fig 3 nal enzymatic activity ofsimple entrapment methods. 1 08 08 07 as 05 os 03 02 04 ° ate ‘=Reamtemperatre ‘Absorbance LbLabginateLbLalginate-LbLalgnate- Hyaluronic Hyaluronic PUS-hibsan Are aci- chitosan’ pojsine PEL acdhosan -acdPEL htoson Fig Intl enzymatie stv of LLentrapment methods. methods, showed high inital activity, unfortunately deactivation Longest stability was observed for LbL sodium alginate-PEI 60% progressed very fast. Highest initial activity from all the methods after 20 weeks (4°C). tested was observed for Lbl sodiumalginate-chitosan, Thismethod Covalent linkage on paper is extremely inconvenient, requires retained more than 50% of initial stability after 10 weeks for stor- multistep preparation and itis almost impossible to wash out the age in 4°C but only for 3 weeks when stored in room temperature. chemicals after reaction. Chemical residues can cause interference EW. ery, LT Kubo Jounal Pharmacerel and rdia nabs 17 (2006) 55 Covalent linkage methods Absorbance toc @Roomtenpertwve Fete i uoG 4k e au ' : _ a : : os on ‘Absorbance PEleapaues CMC-Aljnate Alistecap Sea ‘htosan —_Chtsan- ‘giate Fig 6 nial enzymatic activity of encapsulation methods in posterior applications or slowly deactivate the enzyme as it ‘was seen in case of glutaraldehyde crosslinking. Some treatments (oxidation with periodate, prolonged submersion etc.) alfect the Physical properties of paper, making it easier to tear, ot compro- mising its flexibility. If despite this covalent linkage would be the method of choice EDC/NHS presented best results in this group. It retained 50% of activity after 8 weeks of storage at room tempera- ture. Capsules produced in our laboratory by the syringe method ‘were rather big: sodium alginate ~1.5 mm; sodium alginate-CMC ~2-2.5 mm; sodium alginate-chitosan ~4 mm. Size of those cap- ‘ules could be diminished by using other encapsulation methods like spray drying, but specialized equipment would be necessary. Chitosan capsules produced by interphase polymerization were smaller 0.51 mm. Small capsules and silica beads did not hold to ‘paper, and in the future could be used to produce bioactive paper ‘when mixed in pulp. After complete drying, capsules changed their form becoming flat and completely covered the surface of paper. Inconsistent results were found for encapsulation in alginate with much higher initial activity for storage in room temperature. This can be explained by the incapability to relate the activity with ‘quantity ofenzyme present in each sample in caseof not evenly dis- persed capsules, high standard deviation was also observed in this «ase, Method of choice would be encapsulation in sodium alginate- CMCasit retained more than50% of activity after8 weeks of storage in room temperature, 43.2. Michaelis-Menten kinetics ‘Table 3 summarizes the calculated values of Vinge and Km for {ree and immobilized GOx. Only methods considered most promis ing were tested in this study. All immobilization methods shoved increase in the Km value as compared with the free enzyme. It can be related to the lower accessibility of the active sites of Tables ‘inti parameters fo free and immobiied GO Kenfea)——Van (te) insolation 29 a7 ‘Adsorption tt a8 BSA blocking 158 a8 Entrapment in PVA 106, 49 Enuapmentia starch 10 sa sodium alginate~chtosan 126 “6 Lb iyluronicaid—Pet 0 St Covalent inkageEDCINAS Bi 52 ssa EW. Nery, LT: Rubra /Joural of Pharmacare and medical Atay 1172016) 51-559 immobilized GOx. Lesser Km value indicates higher affinity of ‘enzyme and substrate, which results in faster response, The low- fest increase was noted for EDC/NHS method (28 times higher) ‘which could be explained by the fact that covalent linkage meth- ‘ds tend to specificorientation of the enzyme. Entrapment in starch and BSA blocking showed the highest increase of Km (6.6 and 5.4 times, respectively). Optimization of the amount of BSA and the ‘concentration of starch should be considered in those cases, as to retain their impact on stability but reduce the accessibility effects. Vinax describes the velocity with which the substrate occupies all active sites ofall the enzyme molecules, Decrease of Vix in all ‘cases shows that no simple inhibition is taking place (competitive inhibition—Vmax remains constant, uncompetitive inhibition—Vinax ‘decreases but Km remains constant, acompetitive inhibition—oth Km and Vinay decrease), nevertheless mixed inhibition effects can stil be present, 4, Conclusions In order to be applicable in real-life situations in resource lim- ited settings, where devices can be exposed to higher temperatures ‘during storage and transport (due to i.a. power outages), proposed systems should be designed for enhanced storage stability. It is ‘widely known that adequate immobilization method can signifi- cantly prolong shelf-life of sensors based on enzymatic reactions. ‘One universal immobilization method optimum forall applications does not exist, but basing on this study, some general trends that ‘willheip (0 indicate an adequate method can be observed. No meth ‘ods showed satisfactory results after 24 weeks of storage but the storage stability in 4°C could be enhanced from 8 week (>50% of initial activity) observed for simple adsorption to 20 weeks (Layer by layer sodium alginate-PEN), Storage stability at room tempers- ture increased from 1 week (simple adsorption) to 14 weeks when ‘enzyme was entrapped in starch, It was also noted that protective agents such as sugars, that are Widely used to prolong shelf life of enzymes stored in solu tion have no positive impact on storage stability of glucose oxidase ‘on paper, instead blocking agents such as glycine or BSA should be used (Table 2). All layer-by-layer methods presented consid- erably higher initial activity for the same enzyme loading. Second highest results was noted for adsorption methods and later entrap~ ‘ment. Except EDC)NHS method immobilization by covalent linkage resulted in extremely low initial activity. On the other hand sim- ple entrapment methods and use of blocking agents reduce enzyme accessibility highest increase of km), All methods except of entrap- ‘ment in dextran (14 weeks, °C, >75% of initial activity) and use of BSA as a blocking agent (10 weeks, 4:C, >75% of intial activity), presented rapid decline of initial activity, which usually could be Fetained on a level of 50%. More detailed studies should be cat- fied out with LDL methods including blocking agents such as BSA in subsequent layers, in order to obtain high activity and prolonged ‘stability. Almostall methods enhanced the storage stability inroom temperature, which proves the need of inclusion of immobilization step when fabricating paper-based devices. Acknowledgments ‘The authors would like tothank Fundagao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and National Institute of Science and Technology in Bioanalytics(INCTBio) for financial support, References [1] EW. Nery. LT. ubota, Sensing appraches on pape-hased devices: review. ‘Ama Boaial Chem. 405 2013) 7573-7505, 12] santiago, EW, Nery, GP, Santos, LT Kubota, Mote paper-based ‘beviees oe anaiyalappations. Banas 6 (2014) 89105, 1B) AicYetisen Ms loam, CR Lawe Paper based microldc point-of-care Alagnostcdevies, Lab Chip 13 (2013) 2210-2251 (Ul U- Cai. Huper-Kocure,. Wojceszfska. Immobilzation a strategy fer improving enzyme properties application to exdareductases, Molecules 19.@014)985-0018 (51 MLS Steiner A Duerkop.0.. Wolfs Optical methods for sensing goose (Chem, Soe- Rev 40 (2011) 4805-4838, (61 JD-Nownan, APE Ture, Hore blood glucose biosensors: a commercial perspective, Bosens. lolccon 20 (2005) 2435-2453, (1 MSP Linen, lier Caarcon per Re, Orsanic phase nase erodes ional g. 23 (2006) 135-147. (81 Fong VF Ha Biomelecle immobilization techniques or biosetive paper [Eaton Ana Bioanal Chem. 403 (2012) 7-13. (9) Ciara [D.Breonan. Blsctive paper biomoleileimmobilzation methods Sid pplctons in environmental monitoring, MS Bull 382013) 31-394 (10) Carrio, AW. Martinez, GM. Whitesides, Understanding wax printing Simple mieropatternng process for paper based microfues, Aa Chem 81 (Goo) 7001-7005 U1] MRendl A Bnisch A Mader, K Schuh, 0, Pucker, Brandsteter, he, ‘Ste one step process for nimobiization of iomaleces on payne Substrates based on surfaceatached polymer networks, angi 27 (2011) Giiee2 [12] R-dha.D. Wang, 2 He ¥.Ni, Development of cellulose pape testing tis for altel ase sngchremogen tn ob Pa 8 113] NM Zanon, ON Overs. L Cast, Immobilization of uicasin Langmuir and Langmul- Blodget fs of fay aes and a possible ure cd slrimetnc sensor) Coll nterace S373 (2011) 69-74 [4] | Otonen, Letinen Ero, Feograpicly prised ide structures ia ape, Anal Chem 423010) 1246-10250. U5] Eee, The see encapslation of enzymes, oat. transform. 22 (@o0a) 145-170 [16] EA Shelton Eneyme immobilization: he quest for optimum performance ‘Ads. Synth aa 40 (2007) 1289-1307, 171 W: Banach 0, Chalapakl. CS. Henry. Use of mukpiecolorimetsic Indkeatr for paper ased mirth devices, Anal Chim et. 674 (2010) 207-233, [18] KM Schiling AL Lepore}. Kurlan AW. Martinez Fully encased mueofudi paper-based analytical devices Anal. Charm, 84 (2012) 1579-1585 {19} L MutbacterK. MeGeeney, spas Szabo, V. Leer MA Mateescu Meafied high arylse starch fr immobization of urease for therapetic pplication, Bltchnol App Biochem. 35(2002) 163-170, (20) D brady. jordan, Advances i eneynse nmblsation, Boechaol. Let 31 (@oos) Yeso-nes0 {211 0.Uu. A Mice X Yo, Immobilization and bioactivity of lucose oxidase in Idol mcospneres formulated by an emubsieation-internal eation-adsorpton-polyelecteoyte coating method In. Pat, 339 (Goon) tas-156 {22} ¥-zhang Zs, Zhang, Huang, H. Chea, mmobiization af earbonic yas by embedding and ovalent coupling ino nanocomposite hyaeagel ontaining Hydrate, Polymer (Gul) 30 (2000) 3693-5700, 123] KWo. MALE Cho, spongiform immobilization rctectar of onatropy Poymer hydrogel coentrapping alcool oxidase and horseradish pronase ‘tth octadceaia for opal sensing alco nga sent, Ana (Chen 75 (20) 2270-28, [24] SI Alas M. Onatsia,s. Andrescu, Colorimetric pape bioassay othe

    You might also like