Lab-on-a-Membrane Foldable Devices for Duplex Drop-Volume

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Lab-on-a-Membrane Foldable Devices for Duplex DropVolume Electrochemical Biosensing Using Quantum Dot Tags Christos Kokkinos, Michailia Angelopoulou, Anastasios Economou, Mamas Prodromidis, Ageliki Florou, Willem Haasnoot, Panagiota Sotirios Petrou, and Sotirios Elias Kakabakos Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01625 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016

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Analytical Chemistry

glassy carbon by the addition of high concentrations of Bi(III) (1250 mg L-1) or Hg(II) (10 mg L-1, much higher than the maximum contaminant level of 2 µg L-1 set by the US Environmental Protection Agency for drinking−water) in the sample.26 Hence, these multi−analyte biosassays are not suitable for on−site applications since they require large−volume cells and a complicated assay workflow (i.e. stirring, necessity for extra plating solutions), as well as specific measures addressing the disposal of liquid waste. Therefore, it is highly desirable to develop a new generation of analytical devices for on−site multi−analyte biosensing that could address the aforementioned fabrication and operational drawbacks of the existing ASV−QDs−based assays. In this work, a new type of integrated lab−on−a−membrane foldable electrochemical device has been designed and developed for duplex biosensing using Pb− and Cd−based QDs as labels. The principle of the duplex bioassay and photographs of the device are illustrated in Figure 1. The devices are entirely fabricated by screen−printing onto a nylon 66 membrane and enable the parallel implementation of two different complete bioassays. Each device is composed of two circular assay zones (defined by dielectric ink) which are located symmetrically on either side of a three−electrode screen−printed voltammetric cell (Figure 1B). Two thin lines made of dielectric ink, placed between each assay zone and the voltammetric cell, serve as folding guides (Figure 1B). The voltammetric cell features a graphite working electrode loaded with bismuth citrate (serving as a bismuth precursor), a graphite counter electrode, a silver pseudo−reference electrode and an overlay dielectric layer. Drop−volume bioassays are conducted within each assay zone using antibodies labeled with Pb− and Cd−based QDs, respectively. After acidic dissolution of QDs, the assay zones are sequentially folded one on top of the other along the two folding guides and are attached to the electrochemical cell as illustrated in Figure 1A. The Pb(II) and Cd(II) released from the QD labels are determined simultaneously in a single run by ASV. At the same time with the electrolytic preconcentration of Pb(II) and Cd(II) on the working electrode surface, the embedded bismuth citrate is also reduced in situ to metallic bismuth nanodomains. It is demonstrated that the proposed lab−on−a−membrane devices have numerous advantages, in terms of fabrication and operational characteristics, compared with existing electrochemical paper−based and ASV−QDs multi−analyte biosensors.6−15,19−24 The potential of the presented membrane devices for the analysis of real samples is demonstrated through the development of a duplex competitive electrochemical immunoassay for the simultaneous determination of bovine casein (CN) and bovine gamma−globulin G (bIgG) in milk samples (Figure 1A). The electrochemical devices are further evaluated with respect to their potential for the detection of adulteration of goat milk with bovine milk. The determination of CN and bIgG, as well as the detection of milk adulteration, are of great importance for both the food industry and consumers.27−30

charged nylon 66 membrane modified with quaternary ammonium groups, 0.45µm pore size, thickness 150 µm), used as support for the fabrication of the devices, is from Pall and is cut in pieces measuring 7 cm × 6 cm with the aid of a paper guillotine. Bovine k−casein (k−CN), bovine casein (CN), bovine gamma−globulin G (bIgG), bovine serum albumin (BSA), rabbit polyclonal anti-bovine IgG antibody (affinity purified), 2,2'−azino−bis (3−ethylbenzothiazoline− 6 −sulphonic acid) (ABTS), as well as anti−mouse and anti−rabbit IgG−HRP conjugate are from Sigma−Aldrich. Mouse monoclonal antibodies against bovine k−CN (Mab clones 33−4G10, 33−6Α10) are provided by RIKILT (Wageningen, The Netherlands). Flat−bottomed microtitration wells are obtained from Greiner Diagnostic GmbH. A portable potentiostat PGSTAT101 (Metrohm Autolab) is used for the voltammetric measurements and the baseline correction of the voltammograms is carried out with the NOVA 1.8 software (Metrohm Autolab). The cable connector to the potentiostat was purchased from Dropsens (Code DRP−CAC). Milk samples and protein calibrators. Pasteurized bovine milk (n=2) from different milk companies (DELTA FOOD S.A., Olympos Larissa Milk Company S.A.) are obtained from local supermarkets. Raw goat milk samples are collected from farms located at different areas of South and Central Greece (Attika, Thessaly, Peloponnese). Sodium azide (0.05%, w/v) is added to all samples which are stored in 10 mL portions at -20 o C for up to 6 months. The samples are brought to room temperature and homogenized with sonication for 15 min prior to analysis. The calibrators for the determination of the two analytes (bovine CN and bIgG) are prepared in 20 mmol L-1 phosphate buffered saline (PBS), pH 7.0. The calibrators for the detection of adulteration are prepared by adding appropriate amounts of bovine milk in goat milk.

EXPERIMENTAL SECTION Reagents and Apparatus. Streptavidin (STV)−modified CdSe/ZnS QDs (QD 585 STV conjugated, 1 µmol L-1) are obtained from Life Technologies. STV−conjugated PbS QDs (0.43 µmol L-1) are synthesized and characterized according to published work.31 Τhe nylon membrane (Biodyne B; positively

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Figure 1. (A) Schematic illustration of the duplex protein detection, including the biofunctionalization of the assay zones, the steps of competitive immunoassays and the ASV detection. (B) Photograph of both sides of the membrane and of the PVC holder along with the hydrophilic cottron tabs used for the collection of the liquid waste. (C) Photograph of the complete device (with the membrane mounted on the PVC holder) plugged into the cable connecting to the potentiostat.

Fabrication of the membrane device. Devices are fabricated onto the nylon membrane using a semi−automatic screen printer (DEK 247), stainless steel screens (230 mesh, emulsion thickness 13 µm) and a 75 durometer polyurethane squeegee. The whole fabrication process includes six layers, which are printed in the following order: i) the rings defining the circular assay zones, along with the lines serving as folding guides, are printed with dielectric ink (D2000222D2, Gwent) on one side of the nylon membrane and are allowed to dry for 1 h at 100 o C, ii) a mirror image of the same pattern is also printed with dielectric ink on the reverse side of the membrane and is allowed to dry at 140 oC for at least 5 h, iii) a layer made of silver (PF−410, Acheson) comprising the conductive track for the working (WE) and the counter (CE) electrodes and the reference (RE) electrode, iv) a graphite layer (PF−407A, Acheson) comprising the working and the counter electrodes, v) the bismuth layer (9.4 g graphite ink + 0.6 g bismuth citrate) over the active surface (4 mm diameter) of the working electrode, and vi) a dielectric layer which isolates the conductive tracks of the three electrodes. Each of the layers (iii) to (vi) is allowed to dry for 30 min at 80 oC. Photographs of the devices are provided in Figure 1B, C. Both immunoassays, are performed with the aid of a PVC holder incorporating two wells, located exactly underneath the

assay zones (Figure 1B,C). These wells accommodate highly hydrophilic cotton tabs which are in contact with the bottom side of the assay zones and serve to drain the solutions added in the assay zones. Immunoassays of bovine CN and bovine IgG. A schematic diagram of the biofunctionalization of the assay zones of the membrane device and the competitive immunoassays steps is provided in Figure 1A. For the biofunctionalization, 5 µL of 100 µg mL-1 bovine k−CN and 100 µg mL-1 bIgG solutions in 20 mmol L-1 PBS, pH 7.0 (assay buffer) are placed on each circular assay zone, respectively, and left to dry for 15 min. After the immobilization of proteins, 20 µL of 2.5% (w/v) BSA solutions in 20 mmol L-1 PBS, pH 7.0, containing also 0.05% (v/v) Tween 20 (blocking solution) are applied on both assay zones and left to dry for 30 min in order to block the free binding sites of the membrane. For the immunoassays, 15 µL of a mixture of calibrators in a 1:1 concentration ratio or sample with the respective biotinylated antibodies (250 ng mL-1 from each Mab in case of CN and 500 ng mL-1 for bIgG) is placed on each assay zone and is incubated for 20 min. The assay zones are washed four times with 50 µL of blocking solution and then left to dry for 15 min at room temperature. 15 µL of the appropriate QDs solution (5 nmol L-1 Cd−QDs for bIgG and 4.3 nmol L-1 Pd−QDs for CN


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in assay buffer) is applied to the respective assay zone and the reactions are allowed to take place for 20 min. Finally, the two circular assay zones are washed four times with 50 µL of 0.1 % (v/v) Tween solution in distilled water and are left to dry. ASV measurements. Following the immunoassays, the hydrophobic tabs are removed from the PVC holder and 10 µL of 0.01 mol L-1 HCl solution is placed on each assay zone for 10 min to release Pb(II) and Cd(II) from the QDs labels. Next, the two circular assay zones are folded one on top of the other and attached to the electrochemical cell (Figure 1A). Then, 30 µL of acetate buffer (0.1 mol L-1, pH 4.5) is placed on the exposed (top) assay zone and the preconcentration of the target Cd(II) and Pb(II) is conducted at -1.40 V for 240 s. During the preconcentration step, the bismuth nanostructured film is also formed on the working electrode surface by reduction of bismuth precursor. After the preconcentration step, the potential of the WE is scanned from -1.20 to -0.30 V in the square wave (SW) mode, applying the following conditions: frequency, 50 Hz; pulse height, 40 mV; step increment, 4 mV. Calibration curves for CN and bIgG are constructed by plotting the % ratios of the net stripping current values (corresponding to different calibrators (Ix) after subtraction of the non−specific binding signal) over the zero calibrator current value (I0) vs the protein concentration in the calibrators.

RESULTS AND DISCUSSION Design and fabrication features of the membrane devices. In designing the proposed devices, a number of key objectives were identified: low−cost, high mechanical stability, disposability, high sensitivity using only drop−size sample volumes, capability for duplex biosensing with minimal likelihood of cross−contamination, portability, fabrication and operational simplicity and rapidity, miniaturization, suitability for field analysis and minimal use of external equipment (pumps etc). The final design reflects an effort to reconciliate as many of these attributes as possible and to the highest degree in a realistic device. The proposed devices qualify as lab−on−a−membrane since they fulfill the requirement to perform complete bioassays from sample addition to detection without the need for external equipment other than a detector (portable potentiostat). Regarding the wet chemistry protocol, only additions of drop−size samples and washing/reagent solutions with a micropipette are required. Disposal of the solutions is achieved in a pump−free format, as discussed below in this section. The rationale for the selection of nylon membrane, rather than paper, as the substrate for the fabrication of the complete biosensing devices is to ensure higher mechanical stability during the assays which is well documented.32-35 The Biodyne B membrane is selected, as it can be used without prior activation in contrast to Biodyne A and Biodyne C membranes which need to be preactivated before use for protein binding.35 The Biodyne B membrane is resistant to heat and common solvents and does not shrink, crack, or tear under prolonged use. In addition, in common with paper, it is intrinsically hydrophilic and its porous surface provides a large surface area which facilitates immobilization of biomolecules. In addition, it surface is populated by quaternary ammonium groups which impart a positive charge, enhancing further the immobilization of biomolecules through ionic interactions, without prior chemical derivatization.32−35 Moreover, thanks to the positive charge of the membrane, the released Pb(II), Cd(II) from the

QDs labels are repelled from, rather than trapped in, the pores of the membrane and their mass−transport to the electrode surface is enhanced. A comparative study of the sensitivity between chromatographic paper (Whatman #1 Chr), which is commonly employed as the substrate for the development of paper−based electrochemical sensors and the Biodyne B nylon membrane ia made.6−17 For the comparison experiments, biotinylated BSA (selected as a model protein) is immobilized on both nylon and paper, labeled with STV−conjugated QDs and detected via ASV of liberated cations after acidic dissolution of the QDs labels (the protocol is supplied in the Supporting information). It is found that sharper and 2−fold higher Cd stripping peaks are obtained using the nylon membrane compared with the signal obtained with the chromatographic paper (Figure S1 of the Supporting Information). Furthermore, compared with the existing paper−based biosensors, which are intended for single analyte detection, the proposed devices are designed to permit duplex detection using a single electrochemical cell.6−15 The existence of two spatially separated assay zones enables the parallel implementation two independent assays under different conditions and, at the same time, eliminates problems of cross−contamination. It was decided that the fabrication of the devices was to be carried out entirely by screen−printing (including insulation), in order to avoid more expensive, complex and laborious procedures, such as wax−printing,6−12,15,16 photolithography,17 or vapor−phase silanization,14 commonly used for the insulation of paper devices. It is found that isolation of the assay zones by screen−printing of dielectric ink on one side of the membrane does not lead to effective sealing, as the drops of solutions added diffuse outside the boundaries of the zones (Figure 2Ai). Instead, complete sealing of the assay zones is achieved by a double screen−printing process of dielectric ink on both sides of the membrane and thermal curing at 140 0C for at least 5 h (Figure 2Aii). This thermal treatment does not affect the characteristics of the membrane as confirmed by IR spectroscopy (Figure S3 of the Supporting Information).To facilitate connection of the device to the potentiostat, the silver conductive output tracks for the three electrodes are fabricated to fit to a commercial cable designed as an interface for commercial screen−printed electrodes (Figure 1C). Another practical difficulty encountered is the issue of drainage and collection of the washings/waste produced in the assay zones. To solve this problem, a PVC holder is fabricated incorporating two wells, located exactly underneath the assay zones (Figure 1B). These wells accommodate highly hydrophilic cotton tabs which are in contact with the bottom side of the assay zones and ensure effective drainage without the need of external pumps. Regarding the choice of the core−type of QDs labels, Pb− and Cd−based QDs are selected for the duplex immunoassay since Pb(II) and Cd(II) can be detected simultaneously at bismuth electrodes which excellent sensitivity.36 Typical calibration plots for the detection of Cd− and Pb−QDs using the membrane device are illustrated in Figure S2 of the Supporting Information. In the existing paper−based electrochemical devices,6−17 and in the multiplexed ASV−QDs bioassays,19−24 the working electrodes are commonly made of bare carbon or gold which, nevertheless, impart limited sensitivity for Pb(II) and Cd(II) detection with ASV. Thus, it is imperative that their surface is



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covered with an extra bismuth or mercury layer via complex electroplating approaches to increase the detection sensitivity.13,16,19−24 In contrast, in the proposed devices, the graphite working electrode is modified with bismuth citrate. The bismuth salt in the working electrode serves as an effective precursor for in situ generation of bismuth nanoparticles on the electrode surface enhancing the sensitivity of the ASV detection of Pb(II) and Cd(II), as shown in earlier work.36 Besides, the integration of the bismuth precursor within the working electrode simplifies the assay workflow compared to electroplated electrodes which require the addition of a bismuth plating solution. The generation of bismuth nanostructured film on the working electrode surface occurs during the polarization of the electrode by reduction of the bismuth precursor to metallic bismuth simultaneously with the preconcentration of the released Pb(II) and Cd(II). SEM/EDX analysis of the working electrode surface after treatment at -1.40 V for 240 s, revealed that the in situ formed Bi film consists of Bi nanoparticles (Figure S4 of the Supporting Information). The in situ generation of Bi layer is demonstrated by performing SWASV in the range from -1.0 to +0.30 V. Figure 2B illustrates the SWASV peaks of Pb and Cd released from QDs corresponding to the zero calibrators of CN and bIgG (maximum signal), respectively, and the bismuth oxidation peak without accumulation (dotted line) and after preconcentration at -1.40 V for 240 s (continuous line). Without preconcentration, a weak oxidation peak of bismuth was obtained, due to the oxidation of metallic Bi formed during the anodic scan. Besides, weak stripping peaks of Pb and Cd were observed, due to the short preconcentration at the electrode surface during the anodic scan from 1.0 to +0.30 V. On the other hand, after performing the preconcentration step at -1.40 V for 240 s, a higher Bi oxidation peak appeared, as a result of the larger amount of metallic Bi formed. Stronger Pb and Cd signals were also obtained, since larger amounts of the target cations had time to preconcentrate on the working electrode surface. Development of the immunoassays for bovine CN and bIgG. In the context of this work, immunoassays for the detection of bovine CN and bIgG in milk samples were developed. The detection of CN and bIgG is of great importance for both the food industry and protection of consumers, especially individuals prone to allergies.27−30 Besides, the contents of CN and bIgG in bovine milk are significant indicators of milk quality and play a key role in cheese−making. Several assay parameters, such as the coating concentration of proteins, the blocking solution, the concentration of biotinylated antibodies, the assay buffer, and the duration of different immunoassay steps are optimized. The concentration of k–CN and bIgG used for coating are optimized with respect to the zero calibrator signals of the corresponding assays. It is found that maximum zero calibrator signal is obtained for k–CN and bIgG concentrations of 100 µg mL-1. An important parameter that could affect both the specific and the non–specific binding signal (blank) is the blocking solution composition. Initially, different concentrations of BSA ranging from 0.5 to 5.0 % (w/v) in 20 mmol L-1 PBS, pH 7.0, or in 100 mmol L-1 carbonate buffer, pH 8.5, with or without 0.05% (v/v) Tween 20, are tested with respect to zero calibrator signals of CN and bIgG and the non–specific binding responses. It is found that, by blocking the membrane for 30 min with 2.5 or 5% (w/v) BSA in 20 mmol L-1 PBS, pH

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7.0, containing 0.05% (v/v) Tween 20, the lowest non–specific signals is achieved compared with other blocking solutions tested, while the zero calibrators signal is unaffected. Thus, a 20 mmol L-1 PBS solution, pH 7.0, containing 2.5% (w/v) BSA and 0.05% (v/v) Tween 20 is finally selected as blocking solution.

Figure 2. (A) Photographs of the assay zones after the addition of 50 µL of saturated FeCl3. The assay zone was fabricated by printing of the dielectric ink on: (i) only one side of the membrane, (ii) on both sides of the membrane and thermal curing at 140 0C for 5h. (B) SW voltammograms of Pb, Cd released from QDs (corresponding to zero calibrators of CN and bIgG, respectively) and Bi oxidation peak without preconcentration (dotted line) and after preconcentration at -1.40 V for 240 s (continuous line).

Concerning the antibodies used for the development of immunoassays, they are biotinylated so as to react with the STV– conjugated QDs and their concentrations are optimized. In the case of the CN assay, a mixture of two monoclonal antibodies (at a 1:1 concentration ratio), directed against two different epitopes in the bovine k–CN molecule, was selected according to earlier work. 28,37 For the bIgG assay, an affinity purified rabbit polyclonal antibody was employed, as reported in the literature. 38, 39 Regarding the CN immunoassay, as it can be observed from Figure 3A, when the concentration of antibodies is increased, both the zero calibrator and non–specific binding signal increase. Nevertheless the specific to non– specific signal ratio is optimum when each of the two antibodies is used at a concentration of 250 ng mL-1. Furthermore, the IC50 values (IC50 is the concentration of protein that results in a 50% inhibition of the maximum signal and defines the sensitivity of immunoassay) does not change drastically when the antibodies concentration was increased from 100 to 250 ng mL-1, while it is negatively affected when the concentration of each antibody is increased to 500 ng mL-1. Therefore, a concentration of 250 ng mL-1 for each antibody is selected, providing adequate maximum signal, low non–specific binding signal and adequate sensitivity. Similarly, the optimum anti–bIgG antibody concentration is found to be 500 ng mL-1.


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Several assay buffers are also tested. With respect to pH and buffer composition, it is found that the implementation of a 20 mmol L-1 PBS, pH 7.0, offered both high zero calibrator signal and low non–specific binding as compared with phosphate buffers of equal concentration with pH of 6.5 and 7.4, as well as Tris–HCl buffers with pH values of 7.8 and 8.2.

Figure 3. (A) Effect of the concentration of the biotinylated antibodies (100, 250 and 500 ng mL-1 ) used in CN immunoassay on the response of different CN calibrators (0.0, 0.25, 0.50, 1.0 µg mL-1). In the inset, the bars represent the peak currents corresponding to the zero calibrator and non–specific binding signals obtained using the different antibody concentrations. (B) Effect of duration of immunoreactions between target proteins and the respective biotinylated antibodies on the stripping responses of Pb and Cd released from the respective QDs corresponding to zero calibrators of CN and bIgG. (C) Effect of duration of reactions between biotinylated antibodies and STV–conjugated QDs on the stripping responses of Pb and Cd released from QDs corresponding to zero calibrators of CN and bIgG. Each point represents the average of four measurements at different devices, and the error bars represent the standard deviation from the average.

In addition, the optimum duration of the immunoreaction and the reaction between the biotinylated antibodies with the STV–conjugated QDs is determined (Figure 3B,C). A 20 min incubation time for both steps is selected combining satisfactory signals (higher than 85% of the maximum) with short analysis time. The time–scale of the immunoassay could be reduced by applying shorter reaction times at the expense of sensitivity. Another approach to further simplify and speed up the procedure would be to employ antibodies already conjugated with QDs, leading to a one–step immunoassay. However, this method would necessitate the preparation of dedicated antibody–QDs conjugates specific for each different immunoassay. On the contrary, the STV–conjugated QDs used as labels in this work provide higher flexibility, since they can be used to label any biotinylated antibody and, therefore, the scope of the methodology could be extended to the detection of different proteins. Finally, the effect of the preconcentration time and the preconcentration potential on the stripping response of released Cd(II) and Pb(II) is tested using the respective zero calibrator solutions. The selected values are -1.40 V for 240 s (Figure S5 of the Supporting Information). Analytical characteristics of the duplex CN and bIgG assays. The lab–on–a–membrane devices are applied to the simultaneous ASV determination of CN and bIgG using Pb– and Cd–based QDs as labels, respectively. The analytical characteristics of the developed sensors are demonstrated after performing both assays with different concentrations of proteins (0–5 µg mL-1 for CN and 0–2 µg mL-1 for bIgG). The SWASV voltammograms and the respective calibration plots for CN and bIgG are presented in Figure 4 (panels A, B, C). The coefficients of determination (R2 values) of the curves are 0.995 for CN and 0.997 for bIgG. The limits of detection (LODs) are calculated as the analyte concentration corresponding to the mean net value of the zero calibrator after subtracting three time its standard deviation (SD). Regarding CN, the LOD is 0.04 µg mL-1, while for bIgG the LOD is 0.02 µg mL-1. These LODs are comparable with those reported by other single– analyte electrochemical or optical immunosensors.27–30 The between–sensor reproducibility determined as % relative standard deviation (RSD) using four different devices is lower than 12.2% over the whole calibration range. Determination of CN and bIgG in bovine milk samples. Taking into account the LODs and linear ranges of the duplex assay for CN and bIgG with the proposed devices, their application to the evaluation of bovine milk quality requires different sample dilution for each assay. In particular, the mean CN content of bovine milk is 25.8±1.6 mg mL-1, while for bIgG a mean content of 0.257±0.122 mg mL-1 has been reported, with great seasonal and sub–species variation.40,41 Thus, for CN determination, dilutions ranging from 2×104– to 2×105–fold are required, whereas for bIgG the dilution should vary from 5×102– to 1×104–fold, so that the concentrations of the two proteins in the diluted sample lie within the linear part of the calibration curves. This complication can be readily handled by the proposed devices since the two assays are performed in spatially separated assay zones.



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Figure 4. (A) SW voltammograms for different concentration of CN and bIgG calibrators in assay buffer. (B) The respective calibration plot of CN. (C) The respective calibration curve of bIgG. (D) The calibration curve of goat milk adulteration with bovine. Each point is the mean value of 4 replicates ± SD.

In order to evaluate the applicability of the membrane devices to the simultaneous ASV–QDs determination of CN and bIgG in bovine milk samples, recovery experiments are carried out. For this purpose, two bovine milk samples are spiked with known concentrations of CN and bIgG and then diluted with the assay buffer. For the CN immunoassay, the samples are diluted 8×104–fold, while for the bIgG assay 2.5×103–fold. Besides, the concentrations of two proteins in the untreated samples are also determined with ELISA (Figures S6, S7 of the Supporting Information). The recovery values range from 91 to 108 % for CN and from 92 to 104% for bIgG, while the values determined in the untreated samples are in good agreement with those obtained by ELISA (Table S1 of the Supporting Information). These results confirm the accuracy of the methods developed using the proposed device for the duplex membrane assays. Detection of goat milk adulteration by bovine milk. The detection of goat milk adulteration with bovine is of great interest due to financial reasons. The lower price of bovine milk compared with milk of other species, such as goat, makes it profitable for milk producers to illegally adulterate more expensive milk with bovine milk. For this reason, food companies in the EU and the USA are obliged to state the species of origin of the milk used for production of dairy products.28 In addition, health–related issues arise from adulteration practices, since goat milk is often consumed by individuals with al-

lergy to bovine milk.27–30 Detection of adulteration of goat milk with bovine milk can be performed employing several analytical techniques varying from electrophoretic and chromatographic methods to DNA–based methods.42,43 However, these methods require rather expensive instrumentation, are laborious and unsuitable for on–site analysis. In this work, the electrochemical devices are evaluated with respect to their potential for the detection of goat milk adulteration with bovine milk. Raw milk is a complex matrix containing fat, proteins, and other compounds that could affect the sensor response. Therefore, to avoid matrix effects, goat milk dilutions with the assay buffer ranging from 10– to 100–fold are tested so as to select the lower sample dilution that would not affect the sensor response and at the same time would allow for determination of low adulteration levels (Figure S8 of the Supporting Information). When the goat milk dilution is equal or higher than 50–fold, maximum stripping responses and the lowest non– specific binding signals are obtained for both analytes, thus a 50–fold dilution is chosen. To evaluate goat milk adulteration with bovine milk, curves for CN and bIgG are constructed by plotting the % ratios of the net stripping current values over the zero calibrator current value vs the % (v/v) content of bovine milk in goat milk (Figure 4D). The working ranges extend up to 1% (v/v) bovine milk in goat milk for CN, and up to 50% (v/v) for bIgG; the


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LODs are 0.05% (v/v) for CN and 0.1% (v/v) for bIgG. It is important that the CN assay can detect goat milk adulteration at levels lower than 1% (v/v), while the bIgG assay with its broader working range can determine directly adulteration at levels as high as 50% (v/v), avoiding further sample dilution. This feature, combined with the fact that no pretreatment apart from dilution is necessary for raw milk, is quite helpful for on on–site detection of goat milk adulteration.

CONCLUSIONS This work describes new lab–on–a–membrane electrochemical devices for duplex determination of biomolecules with QDs labeling. The devices are entirely fabricated though a screen–printing process and allow a complete analysis to be performed without the need of external equipment other than a portable potentiostat. The novel design of the devices enables the implementation of two spatially separated bioassays applying different types of QDs signal amplifiers (in this case Pb– and Cd–based QDs). The integration of a bismuth precursor in the working electrode, leading to in situ formation of the sensing layer, permits sensitive duplex ASV determination of released metallic ions from QDs and also simplifies the assay workflow. Furthermore, the devices combine a number of attractive fabrication and operational features such as mechanical stability, disposability, drop–size sample volumes, portability, fabrication and operational simplicity, miniaturization and suitability for on–site analysis. The utility of the lab–on– a–membrane devices was demonstrated for duplex biosensing of CN and bIgG in milk samples and the detection of milk adulteration.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E–mail addresses: [email protected]; [email protected]. Phone:+306972937675. * E–mail: [email protected]. Phone:+302107274298.

Notes The authors declare no competing financial interests.

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Lab-on-a-Membrane Foldable Devices for Duplex Drop-Volume

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