organic reagents and enzymes from zein and

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Delivering inorganic/organic reagents and enzymes from zein and developing optical sensors. Sara Bocanegra-Rodríguez, Neus Jornet-Martinez, Carmen Molins-Legua, and Pilar Campíns-Falcó Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01338 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Analytical Chemistry

Delivering inorganic/organic reagents and enzymes from zein and developing optical sensors. Sara Bocanegra-Rodríguez, Neus Jornet-Martínez, Carmen Molins-Legua, Pilar Campíns-Falcó * MINTOTA research group. Departament de Química Analítica, Facultat de Química, Universitat de València, Dr. Moliner 50, 46100-Burjassot, Valencia, Spain. *Corresponding author: E-mail: [email protected]; Tel.: (+)34963543002; Fax:(+)34963543447

ABSTRACT: Nowadays, the interest for using environmental friendly materials is increasing in many fields. However, the rational design of sensors with biodegradable materials is a challenge. The main of this work is to show the possibilities of the use of zein, protein of corn, as biodegradable and low cost material for immobilizing/stabilizing and delivering different kind of reagents for developing optical sensors. Enzymes, metallic salts, aromatic and small organic compounds were tested. In addition, different techniques of immobilization, entrapped and adsorbed, were used and different formats such as solid devices but also multi-well platform were proposed. Zein capacity for immobilizing two reagents together, enzyme and substrate, into multianalysis format was also shown. Two applications were developed as examples: a colorimetric assay based on ferric hydroxamate reaction for ester drugs, which was applied for atropine determination in pills and a fluorimetric enzymatic multi-well plate biodevice applied to phosphate determination in human serum and urine. Zein demonstrated not to be only a green alternative, but also a versatile polymer for developing sensors from different nature reagents and in different formats and matrices and therefore, applications.

Introduction The main advantages of the immobilization of the reagents for developing sensors are: improvement of stability against environmental factors and over time, easy and fast manipulation for the operator who does not need to prepare the solutions before the analysis and the reduction of the residues generated during the assay. The selection of the optimum support material affects the immobilization process. Several natural materials (e.g. alginate, chitosan and chitin, collagen, gelatin), synthetic polymers and inorganic material (e.g. zeolite, ceramic, silica, glass) have been used as supports for enzyme and other compound immobilization, mainly in other contexts1. There is an increasing interest in using biopolymers to replace and reduce the consumption of conventional polymers derived from petroleum2 such as polyethylene, polydimethylsiloxane (PDMS), acrylics and polycarbonate. Zein is a biodegradable material, a protein of corn composed by a mixture of different peptides, which own several molecular size, solubility and charge3. Alpha zein (22 and 19 kDa) is the most abundant prolamin in corn achieving 71-84% of the total zein content4. Alpha zein has hydrophobic properties5, it can not be dissolved in water but it is soluble in 95% aqueous alcohol or 85% aqueous isopropanol6. This strong hydrophobicity makes zein a unique natural biopolymer and its manipulation has resulted in the development of films, but also gel, coating, micro and nano particles. Although, proteins have been used in some field like alimentary and pharmaceutic industry7-10, not many applications have been performed in the analytical field neither in the sensor fabrication11-15. Zein is mainly obtained as a waste of bioethanol manufacturing. Bioethanol production from corn utilizes dry-grind processing leaving behind protein-rich distillers dried grains with solubles (DDGS) as by-product that later on it is used as low

cost feedstocks. The zein is extracted from DDGS16. The production of bioethanol from corn is increasing over years and therefore, as a by-product, the zein can be obtained in large amounts at low cost. This work investigates whether zein as a biodegradable and low cost material, can provide versatility and advantages for developing optical sensors for several applications. For that, the immobilization/stabilization and delivery of different compounds such as enzymes, metallic salts, aromatic and small organic compounds were tested here. Also, the immobilization of enzyme and substrate in the same support and different techniques of immobilization, entrapped and adsorbed, were assayed and different format such as solid devices but also multi-well platform are proposed. Two solid devices were developed by immobilization of hydroxylammonium chloride and iron chloride in zein films, respectively. The sensors were used to develop a ferric hydroxamate colorimetric assay17,18 as routine quality control analysis of drugs and pharmaceutical products containing ester groups. The characteristics of this assay were compared with those corresponding to the same assay in paper, as a natural polymer, and in PDMS, as a petroleum-derivative polymer; both of them widely used for sensor designs and improved results were obtained by zein. Furthermore, the versatility of zein sensors for immobilizing enzyme and substrate together in an unique solid sensor in a multi-well format were studied. An enzymatic multi-well biosensor fabricated by the immobilization of the enzyme phosphatase alkaline (ALP) and the fluorescent substrate 3-O-methylfuorescein phosphate (OMFP) was developed. The multi-well plate biodevice was used for multianalysis of phosphate in human serum samples. Specifically, the inorganic phosphate concentration in serum has importance in human health because it has a critical role in nu-

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merous normal physiologic functions including energy metabolism, bone mineralization and intracellular signal. The levels of phosphorus in serum are between 3.4 - 4.5 mg/dL according to World Health Organization (WHO). Some pathological conditions could modify the physiological inorganic phosphate concentration in serum19. Urine was also tested with suitable results. Experimental section Reagents. Zein, 3-O-methylfluorescein phosphate cyclohexammonium salt (OMFP), alkaline phosphatase (ALP) from bovine intestinal mucosa (lyophilized power ≥ 10 units/mg solid), glycerol, sodium hydroxide, ethanol were obtained from Sigma Aldrich (Saint Louis, USA). Sodium hydrogen phosphate anhydrous was purchased from Panreac (Barcelona, Spain). Hydroxylammonium chloride was provided by Riedlde Haen (Germany) and the iron(III) chloride hexahydrate from Probus (Barcelona, Spain). PDMS Sylgard®184 Silicone Elastomer Kit(Sylgard®184 silicone elastomer base and Sylgard®184 silicone elastomer curing agent) was provided by Dow Corning (Midland, MI, USA), tetraethylsiloxane, silica nanoparticles (5-15 nm spherical particle size) and ionic liquid 1-methyl-3octylimidazolium hexafluorophosphate 95% were obtained from Sigma Aldrich (Saint Louis, USA). Body fluid was prepared according to20 by using: Sodium chloride (Fisher chemical, Pennsylvania, EEUU), sodium bicarbonate (Probus, Barcelona, Spain), potassium chloride and calcium chloride (Panreac, Barcelona, Spain), magnesium chloride hexahydrate and sodium sulfate anhydrous (Scharlau, Barcelona, Spain), trys(hydroximethyl) aminomethane (Merk, Darmstadt, Germany). Trichloroacetic acid (TCA) was provided from Scharlau (Barcelona, Spain). A stock solution of TCA 10% was prepared. Stock solution of hydroxylammonium chloride (1.79 M) and NaOH (3.14 M) was prepared in ethanol respectively. Stock solution of hydroxylamine (0.895 M) was prepared by mixing the stock of NaOH and hydroxylammonium chloride (1:1) solutions. The stock of iron chloride (0.186 M) was obtained by dissolving an appropriate amount of FeCl3·6H2O in ethanol solution containing 6% of water and 1.6% of HCl (37%). Enzyme stock solution (1 mg/mL) was stored in the freezer until required. Stock solution of OMFP (62.8 µM) was prepared by dissolving (with vortex) an appropriate amount of OMFP in ethanol and it was stored in the freezer. Stock solution of 100 mg/L inorganic phosphate was prepared and different volumes were taken to prepare the standard solutions (up to 5 mg/L). Buffered solution 100 mM was prepared adjusting the pH at 9 with 1 M NaOH. Apparatus. Fluorescence measurements were recorded by a Carry Eclipse Fluorescence Spectrophotometer from Agilent Technologies (Malaysia). The emission was measured at 525 nm inside the white polystyrene 94 multiwell-plate (intact or containing the zein layers assayed) from Sigma-Aldrich (Roskilde, Denmark). UV-Vis measurements were recorded by a HP 8453 UV-Vis Spectrophotometer from Hewlett Packard (USA) furnished with 1 cm path length quartz microcell. Absorption spectra were registered from 400 to 1000 nm. For preparing the standard solutions and the serum samples, Vortex mixer (Labnet International, USA) and Hettich Zentrifuge EBA20 were used (Hettich, Germany).

Determination of ester drugs. Two zein-based sensors were obtained. They were prepared by embedding the reagents iron chloride and hydroxylammonium into zein films. The iron- and hydroxylammonium–zein sensors were prepared by dissolving (10% w/v; 0.16 g of zein) in aqueous ethanol (90%v/v, 1.440 mL of total volume 1.6 mL) along with glycerol (90 µL) as a plasticizer and then the iron (III) chloride hexahydrate (201 mg) or the hydroxylammonium chloride (150 mg), previous grind it with a morter, was added and the mixture was stirred for 20–30 min. Finally, 200 µL for each sensor was placed into a well of a multiwell-plate. After 6h at room temperature, the sensor reagent packages were obtained. The hydroxylammonium-sensors were yellow color while the iron-sensors were brown color. Basic medium is needed for the formation of the hidroxilamine from its salt hydroxylammonium chloride and the formation of the ferric-hydroxamic complex (second step) requires acid medium. In order to adjust the pH in each step, we tested different concentration of NaOH (0.75, 0.5 and 1M) and HCl (0.29, 0.42, 0.5, 0.67, 0.78, 0.89, 1M). Also, we optimized the amounts of the reagents embedded into the zein films for Fe(III)-sensor and the hydroxylamine-sensor. Different amounts of FeCl3•6H2O (25.15 and 9.38 mg/sensor) and NH2OH•HCl (18.75 and 3.75 mg/sensor) were tested. The hydroxylammonium-sensor was added to basic aqueous solution (250 µL, 0.5M NaOH) in an assay tube (pyrex) for 5 min. Then, the sample or standard was added (20 µL) and the solution was heated up to boil. After cooling it down at room temperature, the solution was acidified (275 µL, 0.5 M HCl) and the iron-sensor was added. The solution was centrifuge at 5500 rpm for 5 min and it was measured by UVspectrophotometer at 525 nm. The assay using the reagents in solution was carried out by addition of stock solution of hydroxylamine (90 µL of 0.895 M), the ester compound (0 to 50 µL of 0.1M) and water (300 to 250 µL) into an assay tube and it was heated up. Then, the mixture was cold down at room temperature and it was added the stock solution of iron chloride (300 µL of 0.186 M). The assay on paper was carried out by addition of stock solution of iron chloride (3µL of 0.186 M) and the sample or standard prepared in stock solution of hydroxylamine (10 µL). After 5 min, the paper-based sensor was measured at 525 nm by diffuse reflectance. Calibration curve was prepared up to 85 mM. On the other hand, a PDMS based sensor was also tested. The bulk solid reagent of FeCl3·6H2O (76.4 mg) was directly embedded into the polymer. The solid reagent was mixed with the elastomer base and the curing in a ratio (10:1), but the mixture did not provide a gel. The incorporation of tetraethylsiloxane, silica nanoparticles (5-15 nm spherical particle size) and/or ionic liquid 1-methyl-3-octylimidazolium hexafluorophosphate did not improve the mixture for achieving the gelification of the sensor. Analysis of atropine sulphate in drugs was carried out. Two samples were tested; atropine sulphate drug (1mg/mL) and the Colircusí Atropine 0.5% (which contained 5 mg/mL of atropine sulphate). The experimental process of atropine determination was carried out by adding the sample of atropine sulphate drug (200 µL) directly without any pretreatment in an assay tube pyrex. Also, a solution of NaOH (25 µL of 5 M) and the hydroxylammonium-sensor were added. Then, the solution was heated up to boil. After cooling it down at room temperature, the solution was acidified (30 µL of 5 M HCl)


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Analytical Chemistry and the iron-sensor was added. The solution was centrifuged at 5500 rpm and it was measured by UV-spectrophotometer at 525 nm. Determination of phosphate in serum and urine samples. Two configurations were tested for developing the multiwell plate biosensor :(i) the substrate and the enzyme were entrapped inside the zein film; (ii) the substrate was entrapped and the enzyme was adsorbed on the surface of the film. Enzyme entrapped: 1) The biosensor was carried out by adding 20 µL of a solution (named A) prepared by mixing an aqueous solution of alkaline phosphatase (80 µL of 10 mg/mL), zein in aqueous ethanol (90 % (v/v); 1.6 mL) and 90 µL of glycerol, into the bottom of each well. The biosensor was composed of 1 unit of ALP. After 10 h at room temperature, 20 µL of 4.8 µM OMFP solution (named B) prepared by mixing OMFP dissolved in ethanol to a mixture made up of zein in aqueous ethanol stirred for 15 min in Vortex. 2) The same process was followed but adding first the OMFP, waiting 8 h to complete its drying, and then adding the ALP. Finally, the multi-well plate biodevice was stored at -18°C. Enzyme adsorbed: 1) The biosensor was designed adding 20 µL of the solution B into the bottom of each well. After 8h at room temperature the alkaline phosphatase (10 µL of 0.5 mg/ mL from commercial ALP, 10 U/mg) was adsorbed on the surface of the OMFP zein membrane, obtained from solution B. 2) The biosensor was designed following the same process but adding a second zein membrane on the zein layer containing OMFP. This membrane was synthetized adding 15 µL of a solution (named C) obtained by mixing glycerol (70 % of the zein weight) and zein in aqueous ethanol. After 8h at room temperature, the alkaline phosphatase was adsorbed on the surface of the glycerol-zein layer. Each well contained 0.05 units ALP. The multi-well plate biosensor was stored at -18°C. Standards of phosphate from 0.5 to 5 mg/L were prepared in buffer solution (Tris-HCl, 100 mM pH=9, 200 µL) and were added into each well plate containing the biosensor. The fluorescent measurements were read at 485 nm excitation/ 513 nm emission every 6 seconds for up to 2 min. The fluorescent intensity was plotted versus time. The slope obtained of was the initial velocity for each concentration. The calibration curve of the inhibition of phosphate was calculated from the initial velocity versus the logarithm of the phosphate concentration from 0.5 to 5 mg/L. The assay using the reagents in solution was carried out by addition of OMFP (20 µL of 0.002 mg/mL), inorganic phosphate standard solution (15 µL) and of Tris HCl buffer solution (145 µL, 100 mM, pH=9.0) inside the wells. Then, ALP (20 µL of 0.0125 mg/mL) was thrown in to start the reaction and it was registered the signal as mentioned before. Serum samples stored at -18 ºC in the freezer were analysed. After thawing, the samples were treated with trichloroacetic acid in order to remove the proteins; 100 µL of TCA 10 % was added to 300 µL of serum samples. Then, the samples were centrifuged for 15 min at 3500 rpm. The clear supernatant was transferred to an Eppendorf tube. To carry out the analysis, 15 µL of the treated samples were added to 185 µL of Tris HCl buffer solution (100 mM, pH=9.0). These volumes were placed in the multiwell plate biosensor. 94 experiments can be made. The initial rate was obtained measuring the emitted fluorescence at 525 nm from 30 s to 2 min. The results obtained with the biosensor were compared with the results achieved by using the ammonium molybdate meth-

od.21,22 In addition, urine samples were analyzed. The samples were diluted (1000 x) and the same experimental process described above was followed. Results and discussion Iron- zein and hydroxylammonium-zein sensors. The zein formed stable and functional films following the procedure given in the experimental section, as can be seen in Fig. 1. PDMS and paper were assayed besides zein as supports to carry out the assay of the determination of ester drugs. We tested PDMS as an example of petroleum based material. PDMS-based sensors are easy to manipulate, highly stable and biocompatible 23-26, but not biodegradable. In the case of PDMS-film the solidification (or gelification) of the PDMS was not produced as can be seen in Fig. 1 and then, it is unsuitable for the abovementioned application. The functionality of iron-zein sensor (Fig. 1B) and hydroxylammonium-zein sensor (Fig. 1C) was tested for the determination of ester compounds by ferric hydroxamate formation and spectrophotometry.

Figure 1. Photography of the sensors after being cured for 1 day. A) iron-PDMS sensor, B) iron-zein sensor and C) Hydroxylammonium-zein sensor. The colorimetric ferric hydroxamate assay 17,18 includes two steps; first step is the reaction between the ester and the hydroxylamine to obtain the hydroxamic acid by heating. The second step, is the reaction of the hydroxamic acid with the iron (III) to form the colored ferric-hydroxamic acid complex (Fig. 2A). See experimental section for knowing the development of the assay.

Figure 2. A) Scheme of the hydroxamate assay for ester determination by spectrometry17,18. B) Spectra obtained for (1) propyl acetate 0.1M, (2) cocaine 0.1M, (3) ramipril (0.3 M) and (4) atropine (0.1M). C) Photography of color of the solution after the assay for the calibration curve; (1) propyl acetate from 0 to 3.74 mM (2) cocaine from 0 to 1.81mM (3) Rami-



pril from 0 to 11.19 mM (4) atropine from 0 to 3.67 mM. Heating temperature 100ºC. The spectra registered for different ester compounds: propyl acetate, cocaine, ramipril and atropine at a given concentration are reported in Fig. 2B. The color of the several solutions with increasing concentrations of the mentioned analytes are showed in Fig. 2C. Higher responses of the sensor (higher absorbances and intense color) for propyl acetate and cocaine compared to ramipril and atropine were obtained. Furthermore, the same assay was carried out on paper. Paper is also a natural and biodegradable polymer and it has been widely used for the development of diagnostic sensors and reactive strips. Using paper (Fig. 3), the colors of the reactants (yellow) and product of the reaction (violet-brown) were not homogenous and it was more difficult to discriminate between them compared with the zein-based sensors (Fig. 2). The sensitivity achieved for propyl acetate determination in paper support was lower than that obtained with zeinsensors. In addition, the colour in the paper was not stable over time.

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zein films into the solution and (2) the inhibition of the ALP by phosphate present in solution. In absence of phosphate there is an increase of the signal due to OMFP is hydrolysed by the ALP to form the OMF, which is a fluorescent product. The phosphate inhibits the ALP, therefore the formation of the fluorescent product OMF is slowed down, in other words, the initial velocity of the reaction in presence of phosphate is lower compared to the initial velocity of the reaction without phosphate; see in Figure 5 the line red (in absence of phosphate) compared to grey and black (in presence of phosphate at 0.5 and 2.5 mg/L, respectively). Table 1 compares the initial velocity of both, in absence and presence of phosphate. The initial velocity V0(0) was the velocity of formation of OMF between 0 to 2 min based on the available OMFP in solution and the active ALP (in absence of phosphate). In presence of phosphate the velocity initial decreases as the concentration of phosphate increases (See Table 1, V0(0.5) and V0(2.5)). In order to obtain the calibration curve for phosphate determination; the initial velocity was represented over the logarithm of the concentration of the phosphate up to 5 mg/L. The slope obtained for the different configurations (Entrapped E1, E2 and adsorbed: A1, A2) was reported on Table 1 as velocity or rate constant (K). A P Fosfate

Figure 3. Ferric hydroxamate assay using paper for propyl acetate determination from 0 to 80 mM. Study of the immobilization of both enzyme and substrate in zein. The immobilization of more than one reagent layer by layer in one zein sensor thought immobilization techniques of entrapment and adsorption was studied. The reagent was embedded into the film during its formation (entrapped) or the reagent was deposited on the surface of the film (adsorption). A biosensor is generally based on the immobilization of biomolecules (eg. enzymes or antibodies) in the device.27 The activity of the enzymes is intimately related to its structural conformation which often is highly affected after its immobilization. We demonstrated in a previous paper that the ALP enzyme remained stable and active in zein film and in contact of aqueous solutions released from the film into the solution13. Here, a step further towards greener methodologies by immobilizing both reagents; the enzyme (ALP) and the substrate (OMFP) in one zein sensor and also, miniaturizing and adapting the fluorimetric assay in a well-plate for multianalysis were demonstrated. The volume was scaled from 2 mL to 200 µL (the maximum volume of the well plate) and the multi-well device was tested for phosphate determination in human serum mainly and showed its application in urine. In order to develop the multiwell plate zein-based sensor, several layer configurations (the film disposition into the well plate) and reagent immobilization (adsorbed and entrapped) were assayed into the wells of the plate to obtain reliable results. These designs were based on two overlapping layers of zein with the substrate of the reaction the OMFP and the enzyme ALP, deposited one over the other. In all configurations studied, the OMFP was entrapped in the zein film while the ALP was studied entrapped (E) or adsorbed (A) (Figure 4). The fluorimetric assay for phosphate determination using the zein-based sensor is divided in two steps that can take place simultaneously: (1) the release of the reagent from the

Inhibition

Non-fluorescent substrate OMFP

Fluorescent product OMF

B OMFP

ALP

OMFP

ALP

ALP

OMFP

Entrapped: (E1)

(E2)

OMFP OMFP OMFP OMFP ALP ALP

ALP

ALP

Adsorbed: (A1)

ALP OMFP

ALP

OMFP OMFP OMFP OMFP ALP ALP

ALP

ALP ALP

(A2)

Figure 4. A) Scheme of the fluorimetric assay based on inhibition of ALP in presence of OMFP13. B) Different configuration tested; (E1) ALP entrapped and in the bottom, (E2) ALP entrapped and in the top, (A1) ALP adsorbed on the OMFP sensor and (A2) ALP adsorbed on the glycerol layer. We, first, studied the release of the reagent immobilized by entrapment in zein film for two configurations E1 and E2 by measuring the increment of the intensity over time in absence of phosphate (Figure 4, E1 and E2 in red). As it can be seen in the graphic, the E2 showed higher release compared with E1. Also the linearity was better in E2, so for E1 the intensity of fluorescence started to increase after 1 min. This response was probably owing to ALP in E1 was not able to go easily through OMFP sensor due to its high molecular weight. We avoided the distance of the ALP to the solution by placing the ALP membrane onto the OMFP membrane in E2 (See Fig. 4, E2). However, when we tried to inhibit the ALP in order to quantify the inorganic phosphate, configuration E2 presented problems to quantify the different concentration of phosphate. The inhibition effect was not observed. This is because both


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Analytical Chemistry process, the release (which increase the available active ALP and OMFP in solution which reacted to form the fluorescent OMFP) and then, the inhibition by phosphate, are almost overlapped (In Table 1 the initial velocities for E2 are almost equivalent). To confirm this hypothesis, the sensor was incubated during 30 seconds in a buffer solution. The resulting solution was placed in an empty well and phosphate solution was added. The velocity constant obtained in this case was 26.0 mg L-1 min-1, this was due to the concentration of ALP and the OMFP initial remained constant during the measure (see Table 1, E2’).

Figure 5. Optimization of the multi-well biosensor. Study of the release (in red) and inhibition by phosphate at 0.5 and 2.5 mg/L (in black and grey respectively) for different configuration of the sensor. Table 1: Study of layer configurations to develop the multiwell biosensor for phosphate determination. ALP

Configuration

Solution Free

Entrapped membranes E1 E2 E2’ Adsorbed

A1 A2 A2*

V0(0)

V0 (0.5)

V0 (2.5)

K

109.6±0.9

76.6±0.4

34.6±0.8

-54.3±1.6

94±5

85±5

63±5

-31±2

49±5 129±9

40±5 107±20

30±2 102±17

-13.4±1.1 -10±2

64.6±1.7

42±2

27.8±1

-26.8±1

87±3

74±4

61±2

-17.9±1.6

58.6±1.1 66.5±1.4

47±4 56.0±0.6

35.5±1.5 39.6±0.9

-18.6±1.2 -23.6±0.8

*Using body fluid. E2’: the E2 sensor was incubated 30s and the solution was placed in an empty well and measured in absence and presence of phosphate. Vi(0), Vi(0.5), Vi(2.5) initial velocity in min-1 units obtained for 0, 0.5 and 2.5 mg/L of phosphate, respectively. K is the rate constant in L·mg-1·min-1 units, the slope of the calibration curve obtained by representation of the initial velocity agianst the logaritm of the concentration of phosphate. Only for free membranes 2 mL was employed

As it can be seen, for the configuration in which the ALP was adsorbed on the surface of OMFP membrane (A1), the measured initial velocity (Vi(0)) obtained was higher than that achieved when the enzyme was entrapped in E1. Based on these results, the best configuration so far was the A1; the adsorption of the ALP onto the OMFP membrane. However, it was observed that during the storage of the sensor (thawing process) there was ALP/OMFP reaction, thus the initial velocity was higher. This problem was solved by adsorbing the ALP on a zein layer with 70% glycerol (w/w of zein) situated on the top of OMFP sensor, See configuration A2. The glycerol is a natural plasticizer that demonstrated to increase the porosity of the zein films13 to facilitate the ALP release. For the A2 configuration, the sensitivity was good enough to phosphate determination using the multi-well device at ten

times lower volume (200 µL) than that used for free membranes (2 mL). We showed the sensitivity for A2 configuration was better for body medium, probably due the presence of ions that activate the enzyme 28,29. Therefore, the biodevice with A2 configuration was used for determination of phosphate in human serum. Analytical parameters and stability. The analytical parameters of colorimetric ferric hydroxamate assay for compounds containing ester group are reported in Table 2. Standard solutions for each compound were analysed in triplicate and the average value was used to construct the standard curve using linear regression. The calibration curves were carried out using sensors synthesized in different batches showing equivalent results. The calibration curves were calculated from the absorbance at 525 nm measured against the concentration of the analyte (mM) and are summarized in Table 2. According to the analytes tested, the sensitivity was higher for propyl acetate and cocaine (0.1 mM) compared to atropine (0.4 mM) or Ramipril (0.11mM). The results for propyl acetate from zein-sensors were compared with the paper-based sensor and the reagent in solution. The zein sensor showed smaller LOD and widely linear range than those obtained by the paper-based sensor and using the reagents in solution (see Table 2). Besides, the precision of the method was evaluated by using sensors synthesized in the same batch and performing the assays in the same day (interday) and different batches in different days (intraday) for testing propyl acetate as target analyte. The relative standard deviation was calculated for (n=3) blanks and (n=3) standards of propyl acetate and the values obtained are reported in Table 3. Table 2. Comparison of the figures of merits obtained for a ester compound (propyl acetate) determination by zein and paper-based sensor and the reagents in solution. Analytical parameters using the zein-based sensor for propyl acetate, atropine, cocaine and ramipril. Calibration equation y = a + b·log x1 Analyte

Support material

Linear range1

Propyl acetate

Zein-based sensors Paper-based sensors Solution Zein-based 2 sensors

Atropine

LOD1

a±Sa

0.05-100 0.05-100

0.47±0.01 0.42±0.02

0.36±0.03 0.23±0.05

0.99 0.99

0.01 0.01

0.84-80

0.31±002

34.2±1.1

0.98

0.25

R

7-33

0.037±0.003

-0.01±0.04

0.96

2.36

0.12-100

0.15±0.02 0.16±0.02 0.17±0.01 0.17±0.01 0.158±0.0009

0.31±0.03 0.23±0.04 0.22±0.02 0.14±0.02 0.17±0.02

0.97 0.97 0.99 0.99 0.99

0.04 0.04 0.04 0.04 0.04

0.196±0.009 0.202±0.007

0.99 0.99

0.01 0.01

0.19±0.01

0.99

0.11

Zein-based 0.02-80 1.01±0.01 2 1.02±0.01 sensors Zein-based 0.36-300 Ramipril 0.053±0.002 sensors 1 Units: mM, 2 Sensors obtained from different batches. Cocaine

2

b±Sb

The assay was carried out against possible interferences such as paracetamol, glucose, lactose, starch and caffeine. Only caffeine showed a response, although the solution changed to red-brownish color at concentration level of 250 mM. The stability was assessed by comparing the results obtained for atropine determination in samples with sensors after storage at room temperature, at 4ºC and at -20ºC for 30 days and with sensors prepared the same day. We showed that the



hydroxylammonium-sensor stored at 4ºC and -20ºC did not produce the colored product. On the other hand, the sensors stored at room temperature were structural and functional stable showed similar results for ester determination than sensors prepared before the assay. Therefore, the sensors were stored at room temperature. Table 3. The Relative Standard Deviation values inter and intra-day of the response for a blank and a standard of sensors obtained by two experiences of the same batch (Batch 1) and between different batches for testing propyl acetate as target analyte. Batch Batch 1 Batch 1 Batches

Blank (n=3) Standard (n=3) Blank Standard Blank Standard

Relative Standard Deviation (RSD, %) Intraday Interday Day 1 Day 2 Day 1 1.2 0.6 4.8 1.6 0.2 5.2 1.9 5.1

10.6 2.4 3.4 2.3 8.5

8.5 2.0 6.9 3.5 8.5

Table 4 summarizes the analytical performances of the developed enzymatic biosensor for determination of inorganic phosphate in serum. The inorganic phosphate measured concentration were linear in the range from 0.3 mg/L to 5 mg/L. Standard solutions were analyzed in triplicate (3 wells were employed for each concentration) and the average value was used to construct the standard curve using linear regression. The limit of detection (LOD), defined as the lowest inorganic phosphate concentration that can be detected, was 0.1 mg/L. Besides that, a simulating body fluid solution (see experimental section) made up of different ions and also its spikes with phosphate standards were measured. Although neither Na+ nor K+ show any effect on the ALP activity, ALP activity is reported that increases in the presence of Mg+2 which is found in the serum.28,29 The results given in Table 4 show an absolute value for the slope obtained from body fluid slightly higher than that achieved with aqueous standards. Table 4. Comparison of the figures of merits obtained for phosphate determination in solution and from the A2 biodevice. Calibration equation y = a + b·log x1

ALP/OMFP Linear range1 0.3-5 Solution A2 Biosensor Standard in buffer 0.3-5 A2 Biosensor Standard in body fluid 0.3-5 1 Units mg/L

LOD

a±Sa

b±Sb

R2

mg L-1

57.5±0.6

-55.5±1.0

0.998

0.1

42.2±0.5

-18.6±1.2

0.992

0.1

49.1±0.3

-23.6±0.8

0.997

0.1

The precision of the method was evaluated by using biosensors synthesized in the same batch and performing the assays in the same day (interday) and different batches in different days (intraday). Relative standard deviation (RSD %) obtained were up to 10 % for all: enzyme free in solution and A2 biosensor using standards in buffer solution or in body fluid.

The biosensor demonstrated to be selective to phosphate compared with the ammonium-molybdate21,23, which suffers for interference from arsenates, silicates, sulfides and oxidizing agents. The biosensor was not affected by other ions such as sodium and potassium. The operational stability of the biosensor was investigated by consecutive measurements over time. The biosensor was preserved by freezing at -18°C. The reaction rate was measured in different days after 1h of thawing. The fluorescence response of the biosensor decreased only by 10% after 60 days. About 80% initial activity had been retained after 3 months. Nevertheless, the lifetime of the biosensor is markedly longer than that achieved in solution option assay. Application of iron-zein and hydroxilammonium-zein sensors to atropine determination in drugs. The atropine sulphate drug (1mg/mL) was used without pretreatment and was added to the basic solution containing the hydroxylammonium-sensor and it was followed the experimental procedure. The concentration obtained was 0.942±0.002 mg/mL for n=3 replicas. Also, colircusí atropina (which contain 5 mg/mL) was analyzed. However, the drug contained other ester compounds such as methyl parahydroxybenzoate and propyl parahydroxybenzoate in concentrations that were not reported by the manufacturer. The results obtained were two and three times higher of the concentration of atropine (5 mg/L); 11.805, 10.115 and 15.555 mg/mL; therefore, here, we calculated the total of ester compounds contained in sample that was 12 ± 3 mg/mL expressed as atropine. Application of multiwell plate zein-based sensor to phosphate determination in human serum and urine. The serum proteins can be precipitated by using several organic solvents30. Ethanol and trichloroacetic acid in different concentrations and proportions were tested. Finally, 100 µL of trichloroacetic acid 10% was used to precipitate proteins in 300 µL of serum. In order to evaluate the accuracy of the procedure, serum samples were fortified with different phosphate concentrations between 0.5 mg/L and 2.5 mg/L (Calibration curve obtained: Vi = -23.4 Log [phosphate (mg/L)] + 49, R2=0.997). The recoveries obtained using the body fluid calibration graph were higher than 90 % in all cases, and then matrix effect is absent. A confirmation study was carried out by applying the ammonium-molybdate method21,23. Molybdate method has been commonly used for phosphate determination in water but the reagent is carcinogenic31. Here, we proposed an alternative method that does not use toxic reagents. Table 5 summarizes the concentrations of inorganic phosphate estimated in serum with our method and the ammoniummolybdate method. The concentrations obtained were statistically similar (paired test t, p-value >0.05) to those obtained by the proposed method, thus, the A2 biosensor was able to quantify accurately the inorganic phosphate in serum . For all samples except M1, the phosphorus concentration in serum was between 3.4-4.5mg dL-1 (0.81 to 1.45 mM) according to the blood phosphorus levels by World Health Organization. The proposed biosensor is a versatile device that allows multi-analysis of phosphorus with accuracy in serum. The achieved figures of merit are similar to those reported for single biosensors proposed recently32,33 with the advantage of being a safe and green multianalysis biodevice. In addition, the biosensor was tested in urine of some healthy volunteers to prove its practicality in other matrices. The urine was diluted (1000 x) to be in the linear range of phosphate concentrations and no matrix effect was found.


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Analytical Chemistry Recovery experiments were performed spiking the urine samples with phosphate at 1.5 and 2.5 mg/L; the recovery values obtained were: 100 ± 9 and 98 ± 13 % (n=3), respectively. The concentration found was 570 ± 50 mg/L in agreement with those reported in the analysis of urine of healthy individuals. 34,35 Table 5. Confirmatory study of the multiwell plate A2 biosensor vs ammonium-molibdate method. Concentration of phosphorus estimated in serum samples. Samples M1

Biosensor A2 Found amount (mg /L) 51± 5

Ammonium Molyddate Found amount (mg/ L) 50± 1

M2

37.5± 0.3

36± 6

M3

40± 2

41± 5

M4 M5

34± 4 36± 2

30± 5 39.3± 0.4

Conclusions Zein is a biodegradable polymer obtained as a byproduct of bioethanol production which it is doubly sustainable. Here, we demonstrated that zein is a good support for reagent immobilization and stabilization to obtain sensors for several reasons: (i) different kind of compounds can be embedded and did not interfered in the formation of the film (as it happen with metallic compounds and PDMS) (ii) the enzyme remains active and stable (iii) the fluorophore did not change its characteristics (iv) more than one reagent of the assay can be immobilized on sensor without reaction between them (v) different films and assay format can be developed according to the specific application and (vi) several matrices can be analysed. Finally, two assays were reported as an example; a colorimetric assay based on ferric hydroxamate reaction for ester drugs and a fluorimetric enzymatic multiwell plate biodevice to phosphate determination in human serum and urine and both assays were applied with success to analyte determination in real samples.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], Fax: +(34)963544436

Author Contributions The manuscript was written through contributions of all authors.

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT The authors are grateful to the Generalitat Valenciana (PROMETEO 2016/109) and Spanish Ministerio de Economia y Competitividad-AEI and EU-FEDER (project CTQ2017-90082P) for the financial support received. N.J.-M. is grateful to Generalitat Valenciana for her postdoctoral grant APOSTD/113/2016 program and SB expresses her gratitude to PROMETEO program for her pre-doctoral grant.

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organic reagents and enzymes from zein and

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