1
Zein as biodegradable material for effective
2
delivery of alkaline phosphatase and substrates
3
in biokits and biosensors
4
N. Jornet-Martínez1, P. Campíns-Falcó*1 and E.A.H. Hall*2
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Departament de Química Analítica , Facultat de Química. Universitat of València. Dr.
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Moliner 50, 46100 Burjassot, Valencia, Spain. Fax:(+)963543447, E-mail:
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[email protected]
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Institute of Biotechnology, Department of Chemical Engineering and Biotechnology,
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University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT. Email
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[email protected] .
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ABSTRACT.
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A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and
13
biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of
14
phosphatase substrates. The immobilization and release of both the substrate and/or the
15
active ALP, in a biodegradable and low-cost material such as zein, a prolamine from
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maize, and in combination with glycerol as plasticizer have been investigated. Three zein-
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based devices are proposed for several applications: (1) inorganic phosphorus estimation
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in water of different sources (river, lake, coastal water and tap water) with a detection
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limit of 0.2 mg/L – compared to at least 1 mg/L required by legislation, (2) estimation of
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ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use
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kits developed are low cost, easy and fast to manufacture and are stable for at least 20
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days at -20ºC, so the zein film can preserve and deliver both the enzyme and substrates.
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25
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Keywords: Biomaterial, Zein, Biosensors, Alkaline phosphatase, Pesticide, Saliva.
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1. Introduction
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Plastics have replaced glass and metals in many areas of use due to their properties
29
such as elasticity, biocompatibility, stability, low cost and easy manufacture. However,
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the majority of them are derived from petrochemicals. In addition, huge amounts of
31
wastes are generated and have to be treated, but it has been shown that recycling has
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failed to provide a safe and environmentally-friendly solution.
33
Biodegradable and biocompatible materials extracted from renewable resources have
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received much interest, since they offer less petroleum-dependent and contaminant-
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causing alternatives than plastics. According to ASTM ISO “Environmentally
36
Degradable Plastics”, biodegradable materials are defined as a material in which
37
degradation results from the action of naturally-occurring microorganisms such as
38
bacteria, fungi and algae (Briassoulia and Dejean, 2010). In this context, zein which is a
39
storage protein isolated from the maize endosperm, has shown biodegradation under
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different environmental conditions (pH, Tº and moisture) (Imam and Gordon, 2002) and
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also, in vivo and in vitro studies demonstrated its use in tissue engineering (Lu et al.,
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2011) and drug delivery (Palakurthi and Paliwal, 2014). Zein consists of a mixture of
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polypeptides, dominated by -zein (~20-25kDa, 70-85%), which is rich in leucine,
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proline, alanine, serine and glutamine, and -zein (~15k-30Da, 10-20%), which also has
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a high cysteine content. This creates an amphipathic structure which combines an
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antiparallel -helical structure from -zein, with the N-terminal driven polyproline II
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(Argos et al., 1982) type structure from -zein, held together by disulfide bridges and
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inter- and intra-chain hydrogen bonding. Despite the polar glutamine and serine, this
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results in a rather hydrophobic insoluble material. Rhys and Dougan, for example have
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shown that hydrogen bonding between the side chain and backbone, associated with the
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polyglutamine sequence causes an insoluble structure due to side-chain/backbone internal
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hydrogen bond formation taking preference over hydrogen bonding with water (Rhys et.
53
al., 2013). Nevertheless, ~50% of the amino acid content of zein is polar and side chain
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interactions provide a potentially well-stabilized environment for incorporation and
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stabilization of other proteins.
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Nowadays, there is an increasing demand to develop in situ devices. In order to find
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green alternatives for on site sensing, not only biodegradable materials are required, but
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also, biosensors have been explored, based on enzyme immobilization or using
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combinations of enzymes like alkaline phosphatase (where phosphate and
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organophosphorus compounds are inhibitors) or pyruvate oxidase (where phosphate is
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cosubstrate) (Villalba et. al., 2009). The main mode of detection in this biosensor research
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has been electrochemical, linked with multi-enzyme systems that produce a better
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electroactive product or current amplification. Nevertheless, the same reagents can be
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used in an optical assay, by changing the enzyme’s substrate, so that a
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fluorophore/colorimetric product is generated. For example, alkaline phosphatase is a
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nonspecific phosphomonoesterase that is competitively inhibited by several analytes.
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Previous studies have used alkaline phosphatase for phosphate estimation developing the
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assay in solution (Coburn et al., 1998; Upadhyay et al., 2015) and also for heavy metals
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and pesticide analysis (Upadhyay and Verma, 2013; Berezhetskyy et al., 2008; Garcı́a
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Sánchez et al., 2003; Mazzei et al., 2004; Prieto-Simón et al., 2006), However, these
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papers have not included assays in the field with real environmental samples/biological
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samples or made the step to an integrated solid-state (and ultimately biodegradable)
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sensor.
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This work takes the first steps to investigate whether devices, composed entirely of
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biodegradable materials, could be developed for biokits and biosensors. The systems
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investigated here are based on alkaline phosphatase (ALP) activity/inhibition in the
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presence of phosphatase substrates, such as 3-O-methylfluorescein phosphate (OMFP) or
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p-nitrophenyl phosphate (p-NPP). The enzyme and substrate were packaged in a solid
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film of zein and delivered to the test solution, so that both ALP and OMFP or p-NPP
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diffused from the zein disk into the solution. The potential applications presented here
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are: (1) inorganic phosphate (Pi) estimation, necessary to control nutrients and
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eutrophication (phosphate levels in water are regulated by the EU through the Urban
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Waste Treatment that underlines the maximum annual mean total phosphorus
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concentration of 1-2 mg/L and the Water Framework Directive 2000/60/EC) , (2) the
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estimation of ALP in saliva, which allows distinction to be made between adult and child
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saliva and (3) the organophosphorus pesticide (OPs) estimation in control analysis of
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commercial formulations. In this instance, a comparative study was performed using the
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conventional ammonium molybdate method to validate the results obtained in water. The
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active ALP immobilization, substrate stabilization and delivery system were studied and
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it was shown that zein can be used for encapsulating/packaging and delivering enzymes
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and substrates to the sample during the assay. These simple, environmentally friendly and
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low cost devices can also avoid the need of time-consuming preparation of fresh
93
substrate/enzyme solutions for carrying out the assays.
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2. Materials and methods
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2.1 Materials
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Zein,
p-nitrophenylphosphate
(p-NPP),
3-O-methylfluorescein
phosphate
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cyclohexammonium salt (OMFP) and alkaline phosphatase (ALP) from bovine intestinal
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mucosa (lyophilized power  10 units/mg solid), were purchased from Sigma Aldrich
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(Saint Louis, USA). While absolute ethanol was obtained from Romil (Cambridge, UK)
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and sodium monobasic phosphate from Merck (Darmstadt, Germany).
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2.2 Apparatus
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All the emission measurements were made in a spectrofluorometer Jasco FP-750
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(Tokyo, Japan) and the absorbance measurements in a spectrophotometer Agilent 8453
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(Palo Alto, USA).
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2.2 Preparation of the biokits
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Protein film casting was carried out by dissolving zein (10% w/v) in aqueous
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ethanol (90% v/v) along with glycerol (0%, 30%, 50%, 70% and 90% on a zein weight
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basis) as a plasticizer and then the enzyme, alkaline phosphatase (Commercial ALP, 10
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units/mg ) was added. Fresh alkaline phosphatase (ALP) aqueous solution (80 μL of 10
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mg/mL) was added and the mixture (1.6 mL) was stirred for 20-30 minutes. Finally, it
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was placed into a well-plate mold with 8 positions, 200 μL was used for each biodevice,
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containing 1 unit of ALP (disk S1). After 6 hours at room temperature, the biosensor
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reagent packages were obtained and were frozen (-20ºC) until further usage. The disks
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S2, S3, S4 and S5 were synthetized following the same process with 70% of glycerol and
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using in each case different volumes of ALP aqueous solution of 10 mg/mL: 70, 60, 40
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and 8 μL for S2, S3, S4 and S5, respectively. The disks S2, S3, S4 and S5 contained
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0.875 , 0.75, 0.5 and 0.1 units of ALP.
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The substrate embedded into the film was also prepared. The substrate 3-O-
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methylfluorescein phosphate cyclohexammonium salt (2.4 μM) was dissolved in ethanol
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and was added to the zein mixture, which is zein (10% w/v) in aqueous ethanol (90% v/v)
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along with glycerol 70% on a zein weight basis. The same experimental process was used
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for p-nitrophenyl phosphate (2.02 M) immobilization in zein. Therefore, three types of
123
disks were prepared: one disk was made of ALP immobilized in a zein film, other disk
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was made of OMFP immobilized in zein film and the last disk was made of p-NPP
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immobilized in zein film. All these reagent disks were stored at -20ºC for 20 days.
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Kit A for Pi estimation in waters contains 2 separate disks (one of OMFP
127
immobilized in zein and the other of ALP immobilized in zein) and the buffer (Tris-HCl
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100 mM, pH = 9.0), The kit B for ALP in saliva contains only one disk (p-NPP
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immobilized in zein) and buffer (Tris HCl 100 mM, pH=8) and the kit C for
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organophosphorus in commercial preparations contained 2 separate disks (p-NPP
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immobilized in zein and the other of ALP immobilized in zein) and buffer (Tris HCl 100
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mM, pH=8).
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2.3 Methods
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2.3.1 Fluorescence measurements
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For free alkaline phosphatase in solution, fluorescence measurements were carried
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out in a vial containing Tris HCl buffer (2 mL, 100 mM, pH = 9.0), alkaline phosphatase
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and substrate 3-O-methylfluorescein phosphate (OMFP) (2.4 μM). The OMFP was
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hydrolyzed by alkaline phosphatase to yield 3-O-methylfluorescein (OMF) that was
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detected at 485 nm excitation/513 nm emission in a quartz cuvette. The fluorescent signal
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was captured every 30 seconds for up to 5 minutes and the fluorescent intensity was
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plotted versus time (see Fig. 1 and Fig. 2), the slope obtained of each line represented the
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initial rate (V) for each ALP concentration. Therefore, initial rate as the variation of the
143
fluorescence intensity (RFI) over time (t), gives V = Δ(RFI)/Δt. The initial rate (V)
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increased with increase the enzyme concentration. All the experiments were carried out
145
in triplicate (n=3). For the calibration of free ALP activity in aqueous solution, a
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calibration curve (V = 2.047·[ALP] + 2.2321, R2 = 0.998) was calculated by initial rate
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vs ALP concentration from 0.25 to 50 mg/L (Commercial ALP used is 10 units/mg). A
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calibration curve of inhibition by Pi was calculated by initial rate vs inorganic Pi
149
concentration from 0.5 to 5 mg/L in presence of 10 mg/L of ALP. Finally, the inhibition
150
efficiency was obtained by the logarithm of Pi concentration vs the percentage inhibition.
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The percentage inhibition (% INH) was calculated as follows:
152
153
%INH 
V  Vp
V
100
equation 1
Where V is the initial rate without phosphate and, Vp is the initial rate with phosphate.
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For the ALP disk, measurements were carried out in a vial containing the enzyme-
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zein film disc and Tris HCl buffer (2 mL, 100 mM, pH = 9.0), followed by OMFP (2.4
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μM) addition after 30 seconds. The spiked and unspiked water samples were prepared in
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buffer Tris HCl (100 mM, pH 9.0) and all the emission measurements were carried out at
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room temperature every 30 seconds for 5 minutes. The emission values obtained were
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processed following the procedure mentioned above. A calibration curve was obtained by
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initial rate vs log of Pi concentration from 0.5 to 5 mg/L. In order to compare the response
161
provided by immobilized ALP with respect to free ALP in solution, the following ratio
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called % relative activity were calculated by using the expression:
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% relative activity = 
Vimmobilize d _ ALP
V free _ ALP
 100
equation 2
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For the kit A which contains the OMFP-disk and ALP-disk the experimental
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process was: the OMFP-disk was added to the buffer solution (2 mL, 100 mM, pH = 9.0)
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followed of addition of the ALP-disk after 30 seconds. A calibration curve was calculated
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by initial rate vs log Pi concentration from 0.5 to 5 mg/L. All the emission measurements
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were carried out at room temperature every 30 seconds for 5 minutes. All the experiments
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were carried out in triplicate (n=3). The same ratio established in equation 1 was
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calculated.
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2.3.2 Absorbance measurements
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For free alkaline phosphatase in solution, absorbance measurements were carried
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out in a vial containing Tris HCl buffer (2 mL, 100 mM, pH = 8), alkaline phosphatase
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and the disk of p-NPP immobilized in zein (2.02 M). The p-NPP was hydrolyzed by
175
alkaline phosphatase to yield p-nitrophenol (p-NP) that was detected at 405 nm. All the
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absorbance measurements were carried out at room temperature every 30 seconds for 5
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minutes and all the experiments were carried out in triplicate (n=3). The absorbance data
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use for B and C kits were processed as fluorescence data of kit A: the variation of the
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absorbance over time, Δ(A)/Δt, was used to determine the initial rate (V) and the
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percentage of inhibition (% INH) for the organophosphorus kit C was calculated from
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equation 2.
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3. Results and discussion
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3.1.1 Fluorescence inhibition of the alkaline phosphatase by phosphate in kit A.
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The Pi kit A for measurement of water is based on the inhibition of alkaline
185
phosphatase (ALP) by hydrolyzing non-fluorescent OMFP to fluorescent OMF and
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inorganic phosphate. The OMFP was selected for this initial study due to its high binding,
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efficient hydrolysis and the simple enzyme kinetics compared to the diphosphate
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substrates. Their product OMF, is fluorescent (λex = 485 nm, λem = 513 nm) and also,
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yellow in solution (450 and 485 nm absorption maxima). Both color intensity and
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fluorescence increase over time (Tierno et. al., 2007). Inorganic phosphate acts as an
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inhibitor of ALP competing with the substrate for the enzyme active site. Since the
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fluorescence intensity is proportional to the product concentration, resulting from the
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hydrolysis reaction, the decrease of the fluorescence caused by Pi inhibition was used for
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monitoring the Pi concentration (See the reactions of the kit A in Fig.1). The sensitivity
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required for Pi detection in water (< 1-2 mg/L as required by the EU Directive 2000/60/EC
196
mentioned above) was achieved by the fluorescence method.
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3.1.2 Alkaline phosphatase detection by p-NPP in kit B.
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ALP catalyzes the non-specific hydrolysis of orthophosphoric monoesters to
199
alcohols. The kit B for ALP in saliva is based on enzyme estimation by the substrate p-
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NPP immobilized in zein film. The ALP hydrolyzed the phosphatase substrate p-NPP
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which is an un-colored monoester to become a yellow colored alcohol, the p-NP
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compound after hydrolysis (See the reactions of the kit B in Fig.1). The increase in
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absorbance is proportional to ALP concentration, therefore it is possible to estimate ALP
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levels by absorbance measurements at ex = 405 nm and also, by the naked eye. The kit
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B was applied for estimation of active ALP levels in saliva samples from both adults and
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children.
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3.1.3 Absorbance inhibition of the alkaline phosphatase by organophosphorus.
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The kit C is based on the inhibition of ALP by hydrolyzing p-NPP in presence of
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organophosphorus: chlorpyrifos, chlorfenvinphos and phenytoin (See the reactions of the
210
kit C in Fig.1). In this case, the decreasing of absorbance is proportional to
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organophosphorus concentration. This kit was applied to chlorpyrifos detection in
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commercial pesticide preparations.
213
214
Fig. 1. Picture of devices and reactions involved in the kits A, B and C. Kit A, B and C
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are based on: alkaline phosphatase (ALP) inhibition by inorganic phosphate (Pi) in
216
presence of 3-O-methylfluorescein phosphate (OMFP) , ALP activity in presence of p-
217
nitrophenyl phosphate (p-NPP) and ALP inhibition by organophosphorus (OPs) in
218
presence of p-NPP, respectively.
219
3.2 Developing the devices: glycerol effect.
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For solutions cast onto surfaces (as used here), Yoshino et al. (2002) report that
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assembly into a film is first driven by interactions with the underlying surface, thence
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evaporation of the ethanol and reorganization of zein in the aqueous overlayer. ALP,
223
present during the casting, is expected to become trapped in the zein matrix, possibly with
224
some change in film morphology.
225
Zein films are generally rather brittle with low water vapor permeability (Xu et.
226
al., 2012). In order to improve the access to substrate and active ALP immobilized in the
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film, a more flexible film with higher water vapor permeability (and higher substrate and
228
analyte permeability) is required. Plasticizers with polar groups like glycerol, sugars,
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polyethylene glycol, will generate numerous hydrogen bonds with the zein polypeptide
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chain (Emmambux and Standing, 2007), reducing the intermolecular forces between zein
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chains and potentially increasing water vapor permeability into the interior of the
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polymer (Xu et. al., 2012, Chen et al.,2014). In this work, different concentrations (l0%,
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30%, 50%, 70% and 90% of weight) of glycerol were tested as plasticizer. Glycerol is
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also biodegradable and it has been used as plasticizer in zein films to improve their
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physical properties, by polymer swelling, as a result of diffusion of water molecules into
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the polymeric film matrix (Mastromatteo et al., 2010).
a
b
c
237
238
Fig.2. The variation of the fluorescence intensity over time was used to determine the
239
initial rate (a) for free ALP at concentrations from 0.25 to 50 mg/L (b) immobilized ALP
240
disk (S1, S2, S3, S4, S5 refer to 50, 43.75, 37.5, 25 and 5 ALP mg/L respectively-
241
assuming the total diffusion of active ALP from the disk to the solution) and (c)
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immobilized ALP (10 mg/L) in a zein film prepared with different amounts of glycerol.
243
All the assays were done in buffer solution (2 mL, Tris HCl 100 mM, pH=9.0) with 2.4μM
244
of OMFP the fluorescence was observed every 30s for 5 min.
245
246
The effectiveness of active ALP immobilization was expressed as a % relative
247
ALP activity (See Section 2.3.1), calculated from initial rates for free ALP and
248
immobilized ALP at different % of glycerol (Fig. 2). The diffusion of ALP in the zein
249
film and the immobilized ALP activity were tested by comparing the fluorescent assay
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using the substrate OMFP in buffer and analyzed every 30 seconds for 5 minutes. The
251
responses obtained from both, free ALP in solution from 0.25 to 50 mg/L (Fig. 2a) and
252
immobilized ALP in zein (disks S1-S5, see 2.2 section for experimental details) assuming
253
the total diffusion of ALP, between 5 and 50 mg /L (Fig. 2b), were compared. The
254
fluorescent signal rate increased with increasing enzyme concentration until saturation,
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the enzyme activity in both cases; free ALP and ALP-biodisk followed a classical
256
Michaelis-Menten kinetics (Fig.2). As expected, the biodisks synthesized with different
257
concentration of ALP showed increasing fluorescent rate according to the amount of
258
enzyme immobilized. However, the initial rate at which the fluorescence is observed was
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lower (Fig. 2b) in the ALP-disk than the free ALP (Fig. 2a) in solution, suggesting that
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all the ALP was not completely diffused into solution or the activity of the ALP was
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decreased due to structural changes caused because its immobilization in zein.
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We showed an increase in the relative activity of ALP (see equation 1) from 2%
263
to 22% with increasing % of glycerol (Table 1). This is consistent with an increase of the
264
film hydrophilicity causing the increase of the ALP diffusion into solution. In the
265
presence of the OMFP substrate the fluorescent intensity increases as a function of the
266
ALP activity. However, an overload of glycerol has a detrimental effect on the overall
267
film physical properties. This has been seen in other studies, as an anti-plasticing effect
268
caused by high glycerol concentration, which can damage the continuity of the polymer
269
network of a composite matrix resulting in a reduced integrity of the film (Wongsasulak
270
et al., 2010). When 90% glycerol by weight in zein was used, the ALP-biofilms are not
271
completely formed and are brittle, so it was decided that these disks were not suitable as
272
a reagent platform. Finally, 70% glycerol was chosen as an optimal plasticizer amount
273
for preparation of an ALP- biodevice, since the ALP diffusion is higher than in the other
274
disks (see Fig 2c at 70%: the signal increase is faster than the other disks). The zein film
275
with ALP immobilized (without glycerol) and the film S1 (with 70% of glycerol) was
276
analyzed by SEM. Figure 3 suggests that the porosity of the selected film S1 (Fig. 3b)
277
may be higher compared with a film prepared without glycerol (Fig 3a), due to stacked
278
channels penetrating through the film. As can be seen from the image, the channels in S1
279
appear to be fully distributed throughout the film, which may improve the diffusion of
280
ALP and explain the increasing relative activity with increasing % of glycerol. The
281
relative enzymatic activity observed for this biodisk (22 %) is comparable with that
282
reported for a sol-gel alkaline phosphatase (21 %) (Garcia Sánchez et al., 2003), but
283
presently we are not able to distinguish between loss in ALP activity and poor access to
284
the enzyme.
a
285
b
286
287
Fig. 3. SEM images of zein disk with ALP immobilized using A) 0% of glycerol and
288
B) 70% of glycerol. The porosity is higher in presence of glycerol.
289
290
3.3 Parameter optimization
291
Optimal parameters for the kits were established: OMFP substrate concentration
292
2.4 µM, p-NPP substrate concentration 2.0 µM and pH 9 (see section S1 in the supporting
293
information, SI)
294
3.4 Applications in real samples
295
Before the analysis of the samples, the enzymatic catalysis was calculated
296
according to Michaelis-Menten kinetics (see section S2 in SI)
297
3.4.1 Kit A: Estimation of inorganic phosphate in water samples
298
The application of the biodisk was investigated for tap water, sea (Caracola beach,
299
code DP007), river (Jucár river, code TJU3) and transition waters (Cullera lake, code
300
TES3) collected at different points along the coast of the Comunidad Valencia area
301
(Spain). The samples were prepared in buffer Tris HCl (100 mM, pH 9.0). The fluorescent
302
substrate was added to the ALP-disk in buffer after 30s and the fluorescence intensity
303
signal was measured. Table 2 shows the concentrations of phosphate found using the
304
ALP-disk compared with those provided by ALP in solution. As can be seen the results
305
achieved by the two methods are in agreement. For all samples, the phosphate
306
concentration was lower than 1 mg/L, according to maximum concentration of phosphate
307
in coastal and surface waters allowed by the Water Framework Directive (Directive
308
2000/60/EC). Also tap water was in accordance with the Urban Waster Treatment
309
Directive (Directive 91/271/EEC, < 1mg/L). The phosphate recoveries obtained from all
310
the samples spiked at 1.25 mg/L and at 5 mg/L were near 100 %, so the matrix effect is
311
absent. The samples were also, analyzed by the conventional ammonium-molybdate
312
method [Harris. D.C., 2001]. The mean concentrations of Pi found by the ammonium
313
molybdate method for samples TJU3, TES3, DP007 and tap water are in agreement with
314
those found by the enzyme methods as can be seen in Table 1 considering that the LOD
315
is 1.0 mg/L for this method. The calibration equation was: I = 0.0411 [Pi] + 0.1976 (n=5,
316
R2 = 0.999).
317
Table 1. Phosphate recovery from water samples by fluorescence inhibition of free ALP
318
and ALP immobilized disk.
Water
Standard
Free
Biodisk
Spiked
Biodisk
Spiked
sample
method
ALP
TJU3
≈1(LOD)
0.8±0.1
0.9±0.1
1.25
2.4±0.2
5
6.5±0.5
112, 98
TES3
nd
0.5±0.1
0.4±0.1
1.25
1.6±0.2
5
5.4±0.5
97, 100
DP007
nd
0.5±0.1
0.4±0.1
1.25
1.5±0.2
5
4.7±0.5
103, 89
Tap
nd
nd
nd
1.25
1.4±0.2
5
4.9±0.5
112, 98
[P]
319
nd-not detected
320
[P]-phosphate concentration at mg/L
Biodisk
[P]
Recovery
(%)
321
322
The samples were also tested by using the kit containing both the ALP-disk and
323
the OMFP-disk. In accordance with the results shown in Table 1, all samples except tap-
324
water, showed response between 0.5-0.8 mg/L of Pi.
325
3.4.2 Kit B: Estimation of alkaline phosphatase in saliva
326
A saliva kit was developed using the p-NPP disk and ALP disk. The sample was
327
collected with the following procedure: The bristle end of swab was placed against the
328
inside of the left cheek and was firmly rotated over the entire inside of the cheek area for
329
at least thirty seconds, keeping against the cheek the entire time. Finally, the bristle end
330
of swab was placed into the vial with 1 mL of buffer Tris HCl pH 8.
331
A standard method was used for ALP quantification in saliva, therefore
332
concentration of ALP of 0, 20, 40, 60 units/L were added to 1 mL buffer with the swab
333
and were measured by absorbance. The calibration curve was obtained by representation
334
of the initial rate vs ALP concentration added for each of eight samples (M1-M8) (n = 3),
335
see Table 2. The samples of children M7 and M8 showed higher concentrations of ALP
336
than adults samples (See Table 2). The ALP concentration is indicative of periodontal
337
tissue destruction (Bezerra et al., 2010) which is associated with teething in children and
338
in some gum conditions. We believe that this method could be applied in forensics to aid
339
to know if salivary sample is from children or from adults.
340
Table 2. Alkaline phosphatase activity estimation from salivary samples by absorbance
341
of p-NPP immobilized disk.
342
Healthy volunteers
343
344
Linearity, y = a + bx (n=5)
Found
Sample
Gender
Ages
b
Sb
a
Sa
R2
M1
Female
22
0.007
0.002
0.065
0.033
0.95
< LOD
M2
Male
23
0.023
0.002
0.001
0.045
0.97
8
M3
Female
22
0.017
0.001
0.035
0.020
0.99
20
M4
Female
26
0.019
0.001
0.044
0.031
0.98
9
M5
Female
27
0.013
0.001
0.013
0.017
0.99
 10
M6
Female
15
0.021
0.002
0.024
0.043
0.97
12
M7
Male
4
0.016
0.002
0.062
0.041
0.96
32  5
M8
Male
2
0.018
0.002
0.042
0.034
0.97
24  5
concentration
y = absorbance, x = concentration of alkaline phosphatase (units/ L) b: slope, S b: standard deviation of the
slope, a: ordinate, Sa: standard derivation of the ordinate, R2 correlation coefficient.
345
346
3.4.3 Kit C: Detection of organophosphorus in commercial pesticide preparation.
347
A colorimetric kit for screening organophosphorus such as chlorpyrifos,
348
chlorfenviphos and phenytoin was studied. The procedure described in section 3.4.1. was
349
followed. The ALP inhibition for chlorpyrifos, chlorfenviphos and phenytoin were 34, 12
350
and 13% respectively. The calibration equation for chlorpyriphos was : V = 0.0015 +
351
0.5542 [chlorpyrifos (g/L)], R2>0.99. The chlorpyrifos concentration, in preparations of
352
a commercial product, was found as 360  20 g/L, which is consistent with the declared
353
360 g/L (Italo/Clorcirin product).
354
This method could be useful for control analysis by visual inspection in pesticide
355
preparations required for pest control in gardens and crop fields. Based on the results, the
356
kit offers a binary YES/NO response for qualitative analysis of chlorpyrifos. The assay
357
can be used as a test for screening of samples with positive or negative response. The
358
relative false positives and false negatives were determined analyzing 10 standard
359
solutions of chlorpyrifos at 50 M and 10 standards solutions of chlorpyrifos at 100 M.
360
The binary responses YES/NO were obtained by absorbance measurements and visual
361
inspection. The kit showed that it is reliable for chlorpyrifos detection at 100 M at 90%
362
of confidence level, which is within the limits established by the law.
363
3.5 Precision and stability.
364
The precision of the immobilized ALP in back to back manufacture was assessed
365
by carrying out the process six times using the same experimental process and the relative
366
standard deviation (RSD) was 6.6 % (n = 6). The precision for phosphate detection at 1
367
mg/L by kit B and chlorpiryfos detection at 100 M by kit C were calculated for n=3
368
measurements for 5 days and the relative standard deviation (RSD) was 6.2 % and 14 %,
369
respectively.
370
The storage stability was assessed by measuring activity after storage at room
371
temperature, at 4ºC and at -20ºC for 20 days. The devices, stored at room temperature and
372
4ºC decrease in ALP activity by about on two-thirds (14%) while for the biodisk at -20
373
ºC, the ALP activity remains unchanged around 21-26%. Also, the substrate disk
374
continued to show sensitive responses when they were stored at -20ºC.
375
4. Conclusions
376
Simple disposable environmentally friendly devices were developed by using a
377
biodegradable material like zein to create an analytical bio-disk intended for a single-
378
use. These bio- disks can avoid the need of preparing fresh reagent solutions of enzyme
379
and/or substrates for carrying out the assays, improving their stability, safety and
380
usability. The disk is designed to be disposable with the enzyme and substrate diffused
381
from the disk into the sample solution. In this work, we showed successful immobilization
382
and delivery of both the substrate and the enzyme. The kits A, B and C were applied to
383
the measurement of Pi in water of different sources (river, lake, coastal water and tap
384
water), of ALP in saliva and chlorpyrifos for control analysis in a commercial product
385
preparation, respectively. In this context, we have described several options for
386
biodegradable kits, that take the first step to respond to the needs of analysis in several
387
fields : in-situ monitoring campaigns in environmental and the use of non-invasive
388
samples in diagnostic tools . The zein biodisk can be crafted in different shapes and could
389
be directly integrated with a measurement device (eg ccd) for simple monitoring, eg via
390
a mobile phone (Villiers et al., 2015; Pla-Tolós et al., 2016).
391
Acknowledgements. The authors are grateful to Generalitat Valenciana (PROMETEO
392
program 2012/045 and BEFPI) and
393
Competitividad/FEDER (project CTQ2014-53916-P). NJ expresses her gratitude to
394
PROMETEO program for her predoctoral grant and BEFPI program for her stay grant.
395
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