Disposable amperometric immunosensor for the detection of

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Disposable amperometric immunosensor for the detection of adulteration in milk through single or multiplexed determination of bovine, ovine or caprine immunoglobulins G Víctor Ruiz-Valdepeñas Montiel, Eloy Povedano, Sara Benedé, Luis Mata, Patricia GalánMalo, María Gamella, A. Julio Reviejo, Susana Campuzano, and José M. Pingarrón Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02336 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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Analytical Chemistry

Disposable amperometric immunosensor for the detection of adulteration in milk through single or multiplexed determination of bovine, ovine or caprine immunoglobulins G Víctor Ruiz-Valdepeñas Montiel†, Eloy Povedano†, Sara Benedé‡,∗, Luis Mata, Patricia Galán-Malo, María Gamella†, A. Julio Reviejo†, Susana Campuzano†,∗, José M. Pingarrón†,∗ †Departamento

Química Analítica. Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain ‡Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), E-28049, Madrid, Spain ZEULAB,

S.L., Bari, 25, E-50197 Zaragoza, Spain

ABSTRACT: This paper reports the first immunoplatforms for the detection of adulteration in milk with milk or colostrum from other animals. The developed electrochemical bioplatforms, allow the reliable determination of immunoglobulins G (IgGs) from cows, sheep or goats. They rely on sandwiching each animal species-specific IgGs with selective antibody pairs (unconjugated and conjugated with HRP) onto magnetic microbeads (MBs) used as solid supports and amperometric transduction with the H2O2/hydroquinone (HQ) system at disposable electrodes. The immunoplatforms allow achieving LODs of 0.74, 0.82 and 0.66 ng mL-1 for bovine, ovine and caprine IgGs, respectively, which are lower than those obtained with conventional ELISA methodologies and in 25 times shorter time. The bioplatforms were successfully applied to the determination of the individual content of the target IgGs in milk samples of different animals (cow, sheep and goat) and type (colostrum, raw and pasteurized), without matrix effect and after just a sample dilution. They were also applied to the detection of adulteration with milks from other animals at levels below than those required by the European legislation (1.0 %, v/v). The possibility to detect milk adulteration with colostrum using a strategy based on the measurement of the total content of the three target IgGs in raw milks is also demonstrated. Multiplexing platforms were constructed to be used in routine surveillance of milk. They are able to provide in a single run and in just 30 min relevant information regarding the milk sample including its animal origin, the undergone heat treatment and whether it was adulterated with milk or colostrum from other species.

Food adulteration affects both the quality of the product and its processing and it may pose health risks and economic and confidence problems for the consumers.1,2 Milk, olive oil, honey, saffron, orange juice, coffee and apple juice are the most common targets for adulteration.2 Dairy products consumed worldwide such as milk, yogurt or cheese are an important part of the diet because of their high nutritional value and have great economic importance in the food industry.3,4 Milk can be adulterated by addition of water, neutralizers to mask acidity, salt or sugar to mask extra water or high solid contents, whey and dairy products of different animal origin. Many substances, most of them toxic, have also been found as adulterants in milk, such as hydrogen peroxide, formalin, octylphenol, nonylphenol, urea, starch, carbonates/bicarbonates, boric acid, melamine or ammonium sulphate,2,4,5 which may involve relevant clinical risks causing vomiting, diarrhea, decreased body temperature, weak irregular pulse, stomach damage (gastritis and inflammation of intestine), decrease sperm production, pain in lower abdomen, renal damage or breast cancer.4 Milk adulteration with milk from different animal species or with colostrum from the same or different animal species is considered nowadays an important problem of great interest

for the protection against species substitution or admixture in dairy products. This fraudulent adulteration must also be avoided due to religious, ethical or cultural reasons and to health-related issues (intolerance or allergy to milk from certain species).3,6-8 Therefore, for legal reasons and for consumer protection and confidence, milk should be authentic and correctly labelled. However, this type of milk adulteration is difficult to detect due to the similar composition among the different animal species.1,7 The prevailing production of cow's milk in the world makes it one of the low-priced food products and it is the main type of milk used for the adulteration of other milk types, such as sheep, goat or buffalo, used in the food industry but with a lower or highly seasonal production, resulting in a deficit in some periods of the year which increases their price.1,3,8 These widespread unlawful practices take advantage of the overproduction and cheaper price of cow’s milk to fraudulently adulterate more expensive milks and cheese prepared from them to avoid loss of profit.3,9 Moreover, the production of traditional cheese such as Roquefort, Feta, Manchego or Buffalo mozzarella in Europe is attaining considerable economic importance, and the European Legislation demands the good labeling of these products stating the milk species used for cheese production or other

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dairy products. It is worth noting that only buffalo mozzarella cheese produced from 100 % of buffalo milk are correctly labeled and sold.6,10,11 Therefore, identification of the animal species from which the milk or cheese are produced is a remarkable challenge, and it is essential to ensure milk and dairy products are unadulterated and accurately tagged at the levels equal or lower than 1.0 % (v/v) adulteration according to the European Community (EC) Regulation.3.8,9,12 On the other hand, the intentional addition of raw colostrum to milk is an unlawful and undesirable practice for the dairy industry, due to the presence of higher whey proteins/casein ratios in colostrum, which alters the organoleptic properties of dairy products and causes technological problems in their production.13,14 Colostrum is the form of milk secreted during the first few days after calving, whose main function is to provide immune protection and nurture the newborn.14 Colostrum has 50 to 100 times higher levels of whey proteins (β-lactoglobulin, α-lactalbumin, immunoglobulins, lactoperoxidase (LPO), lactoferrin, proteinase inhibitors, albumin, α2-macroglobulin and transferrin), vitamins and minerals, and lower levels of lactose and casein than milk.13,14 The presence of these proteins at high concentration can cause their precipitation on the surface of equipment during heat treatments, disrupting technological processes or rendering them less effective. Particularly, in cheese industry, the colostrum presence decreases the manufacturing efficiency due to the low casein levels and the high concentration of lactoferrin and lactoperoxidase, and the non-specific antimicrobial properties that affect negatively the growth of bacteria responsible for fermentation and cheese maturation.14,15 However, there is no legislation clarifying the levels of accepted colostrum in milk, because there is no clear delineation between colostrum and milk. It has been described that after 7 days of lactation (legal time for milk retaining after calving) IgG levels are below 1.0 mg mL-1. Moreover, it has been reported that the addition of 20 % of cow’s colostrum or only 5 % of goat´s colostrum causes technological problems.1315 In addition, the marketing of colostrum is prohibited in some European countries. Due to the non-existent legislation, the use of these values (5 and 20 % of goat´s and cow’s colostrum, respectively) can be considered as cut-off points for milk adulteration with colostrum. Immunoglobulins (Igs) are important components of the immunological activity of colostrum and milk, transported through the mammary epithelial cells and transferred out of the mammary gland by milk ejection. In ruminant milk there are three classes of immunoglobulins: IgG, IgA and IgM. IgG represents 80 % of total Igs, and IgG1 is the predominant subclass.14-18 The IgGs specificity among different animal species and the high levels found in raw milk and colostrum, make IgGs suitable biomarkers to discriminate the animal origin of milk and the best indicator of the presence of colostrum in milk due to the high colostrum/milk IgGs ratio.14,15 Moreover, IgGs are not easily liable to proteolysis and are stronger immunogens than caseins and other whey proteins, which make them very interesting targets for detecting species adulteration in milk.1,3 Several methods have been reported for the detection of milk adulteration focused on studying milk composition from different animal species. These methods include separationbased techniques such as electrophoretic strategies9 (isoelectric focusing which is the current European Commission (EC)

reference method3 and capillary electrophoresis) and chromatographic methodologies (reverse phase highperformance liquid chromatography, ion exchange chromatography1 and size exclusion chromatography17). Nephelometry15 as well as methods involving single frequency conductance measurements, digital image, ultraviolet–visible or infrared spectroscopy2 have also been proposed for this purpose. Special emphasis should be made on immunological or DNA based techniques, with enough sensitivity to detect 0.5 or 0.1 % of milk-specific animal species, respectively.1 Most of the immunological methods reported so far to detect milk adulteration use antibodies targeting casein, lactoglobulin and other whey proteins3,6 or IgGs.9,15,17 The used techniques include immunochromatography,9 radial immunodiffusion,15 lateral flow immunoassay,8 radial immunodiffusion17 or enzyme-linked immunosorbent assay (ELISA), which is the most common method to determine milk IgGs for adulteration identification. Most commercial ELISA kits have detection limits between 0.71.95 ng mL-1 for IgG detection or 0.1 % of species-specific milk (see Table S1). However, they show some limitations including false positive results due to crossreactivity, proteolysis occurring during heat treatment,6 the requirement of multi-steps, expensive instrumentation only available in centralized laboratories and commercial unavailability of ELISA kits for multiplexing detection of different mammals IgGs. In the particular case of lateral flow (immuno)assays, although they do not require multi-steps or expensive instrumentation, they are used mostly for qualitative and semiquantitative analyses and in a much smaller extent for quantitative monitoring.19 On the other hand, DNA based methods are popular for examination of food products, because DNA is stable under high temperatures or pressures used during food product processing. It should be highlighted the use of polymerase chain reaction (PCR) or variants as PCR-Restriction Fragment Length Polymorphism PCR-RFLP7 or species-specific PCR11 for species identification. However, there are still significant problems with the use of PCR for quantification, since these methods are time-consuming, prone to the existence of false negatives,6 and more challenging than immunosensing strategies to be implemented in the decentralized devices that today's society demands to assure food quality, safety and genuineness. In this context, electrochemical biosensing platforms based on the use of immunomagnetic capture have demonstrated excellent performance for the determination of target biomarkers in complex food matrices with minimal sample treatments and using quite simple protocols.20-22 Indeed, the advantages provided by the use of magnetic beads (MBs) in electrochemical biosensing in terms of large area, easy functionalization, rapid assay kinetics, improved sensitivity, matrix effect minimization and easy location/transport control by a magnetic field,23-26 make this strategy particularly adapted for such purpose. In addition, only the MBs are incubated with the sample in the strategies coupling MBs and SPCEs, thus avoiding undesirable (bio)fouling of the electrode surface when complex and protein-rich samples such as milk are handled. Electrochemical immunosensing platforms involving MBs have been successfully employed even for multiplexing of relevant allergenic proteins or antibiotics in food extracts27 and 1:1 diluted milk samples,28,29 respectively, by preparing different batches of MBs modified with the specific antibodies to the targets to be determined.


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Analytical Chemistry This paper reports the first immunosensor for the detection of milk adulteration with milk/colostrum of other animals through the single and/or simultaneous determination of bovine, ovine, caprine or total IgGs contents. The strategy relies on the use of sandwich-type immunoassays using selective capture and HRP-labeled detector antibodies for each target mammal IgG and carboxylic acid-modified magnetic beads (HOOC-MBs). Amperometric measurements (at 0.20 V vs. the Ag pseudoreference electrode using the H2O2/hydroquinone (HQ) system) at single or four screenprinted carbon working electrodes were used for the single or multiplexed monitoring of the affinity reactions and the determination of the target IgG concentration. The immunoplatforms were successfully applied to the sensitive and specific determination of cow, bovine and caprine IgGs and to detect milk adulteration at the level demanded by European Legislation.

EXPERIMENTAL SECTION Apparatus and electrodes.

Amperometric measurements were performed with a CHI812B (CH Instruments) potentiostat controlled by software CHI812B and µStat 8000 Multi Potentiostat/Galvanostat (MetrohmDropSens S.L.) controlled by software DropView 8400. Screen-printed electrodes with one (SPCE, DRP-110, 4-mm diameter) or four working carbon electrodes sharing auxiliary and reference electrode (SPC4Es, DRP-4W110, 2.95-mm diameter) from Metrohm-DropSens S.L. were employed as electrochemical transducers for single and multiplexed determinations, respectively. They were used with the appropriate specific cable connectors (DRP-CAC and DRPCONNECT4W from Metrohm-DropSens S.L.) and homemade Teflon casings with 1 or 4 neodymium magnets (AIMAN GZ) embedded to be located just below each working electrode (see Figures 1c and d) thus ensuring an efficient and reproducible capture of the modified-MBs. All electrochemical measurements were carried out at room temperature. A Bunsen AGT-9 Vortex was used for homogenization of the solutions. A Thermomixer MT100 constant temperature incubator shaker (Universal Labortechnik) and a magnetic stand DynaMag™-2 (123.21D, Invitrogen Dynal AS) were used for MBs incubation under stirring and their magnetic location during the washing steps, respectively. A thermocycler (SensoQuest LabCycler, Progen Scientific Ltd.) was used for the thermal treatments applied to milk samples.

purchased from Gerbu. Polyclonal antibodies selective for bovine, ovine or caprine IgGs were kindly provided by ZEULAB, S.L. Antibodies were affinity purified and used without modification as capture antibody (CAb) or conjugated to HRP as detector antibody (HRP-DAb) for each target IgG. The following solutions were prepared with Milli- Q water: 0.025 M MES buffer, pH 5.0; 0.1 M Tris-HCl buffer, pH 7.2; 0.1 M phosphate buffer, pH 8.0 and 0.05 M phosphate buffer, pH 6.0. Activation of the HOOC-MBs was carried out with an EDC/sulfo-NHS mixture solution (50 mg mL−1 each in MES buffer, pH 5.0). The blocking step was accomplished with a 1.0 M ethanolamine solution prepared in a 0.1 M phosphate buffer solution of pH 8.0. Details about all the protocols used (Bovine, ovine and caprine IgGs immunocaptors, Single or multiplexed detection of individual bovine, ovine, caprine or total IgGs, Amperometric measurements, Analysis of milk samples) are described in detail in Supporting information.

RESULTS AND DISCUSSIONS The fundamentals involved in the preparation of versatile immunoplatforms for the determination of bovine, ovine, caprine and total IgGs are schematically displayed in Figure 1. In brief, three different specific-pairs of antibodies, comprising a capture antibody (CAb) and an HRP-conjugated detector antibody (HRP-DAb), were used for each target IgG. The preparation of the immunocaptors was accomplished by covalent attachment of the specific-CAb antibodies onto carboxylic-MBs, previously activated with an EDC/SulfoNHS solution, and further blocking with ethanolamine (Figure 1a). The target IgG was sandwiched in a single step by incubating the prepared immunocaptors with the standard/sample solution supplemented with the specificHRP-DAb. The MBs bearing the sandwich immunocomplexes (HRP-DAb-target IgG-CAb) were captured magnetically on the working electrode surfaces as described in section Amperometric measurements (in the Supporting Information), and the immuno-recognition events were monitored by measuring the cathodic current at 0.20 V vs. the Ag pseudoreference electrode using the H2O2/HQ system.30 The SPCEs/SPC4E were only used to perform a single measurement and discarded afterwards.

Reagents and solutions. All the reagents used were of analytical-reagent grade, and deionized water was obtained from a Millipore Milli-Q purification system (18.2 M cm). Carboxylic acid-modified MBs (HOOC-MBs, 2.7 μm Ø, 10 mg mL−1, Dynabeads M-270 carboxylic acid, Cat. No: 14305D) was purchased from Dynal Biotech ASA. Casein blocking solution (a ready-to-use, PBS solution containing 1.0 % w/v purified casein) was purchased from Thermo Fisher Scientific. NaCl, KCl, sodium di-hydrogen phosphate, disodium hydrogen phosphate, and Tris-HCl were purchased from Scharlab. N-(3-dimethylaminopropyl)-N’ethylcarbodiimide (EDC), ethanolamine, hydroquinone (HQ), hydrogen peroxide (30 %, w/v), and IgGs from bovine, goat or sheep sera were purchased from Sigma-Aldrich. Nhydroxysulfosuccinimide (sulfo-NHS) was purchased from Fluorochem. 2-(N-morpholino)ethanesulfonic acid (MES) was ACS Paragon Plus Environment

Analytical Chemistry Figure 1. Schematic display of the fundamentals involved in the preparation of the MBs for the determination of single bovine, ovine, caprine IgGs a) or total IgGs b); pictures with the corresponding homemade magnetic holding blocks and SPEs and drawings with the type of MBs captured on the surface of the SPCE c) and SPC4Es d) used to perform the single or multiplexed amperometric determination of animal species-specific IgGs, respectively.

Optimization of the working variables. Figure 2 shows the amperometric responses obtained with biosensors constructed with unmodified MBs and with MBs modified with the specific-CAb for each type of IgG in the absence and in the presence of 250 ng mL-1 of each IgG standard. The obtained results show fairly well the feasibility of the developed strategy for the determination of each target IgG since significant differences in the amperometric responses obtained in the absence and presence of each IgG standard occurred only when the MBs were modified with the CAb specific to the target IgG.

HRP-DAb was made in a single step (panels 1-3a in Figure S2), thus allowing the development of simpler methodologies in a shorter assay time. With the aim of simplifying and unifying the protocol for multiplexed determination, 45 and 30 min were selected as incubation times for the specific-CAb immobilization (panels 1-3b in Figure S1) and the target IgG sandwiching onto the immunocaptors (panels 1-3c in Figure S2), respectively, without compromising the sensitivity significantly.

Analytical and operational characteristics. Calibration plots constructed for bovine 1), ovine 2) and caprine 3) IgG standards are displayed in Figure 3. The corresponding analytical characteristics are summarized in Table 1. As it can be seen, similar linear ranges and sensitivities were achieved for the three IgGs, which facilitates the integration of the biosensors in a multiplexed platform. The limit of detection (LOD) values were calculated according to the 3sb/m criteria, where m is the slope of the linear calibration plot, and sb was estimated as the standard deviation for 10 amperometric responses measured in the absence of the target IgG. The LOD values were 0.74, 0.82 and 0.66 ng mL-1 for bovine, ovine and caprine IgG, respectively. 2000

1) 2) 3)

1500

-i, nA

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1000 500 0

Figure 2. Amperometric responses obtained with biosensors constructed for the determination of the target IgG standards (bovine (B), ovine (O) or caprine (C)) with unmodified MBs (striped bars) or with the immunocaptors prepared for bovine (1), ovine (2) and caprine (3) IgGs. Error bars were estimated as triple of the standard deviation of three replicates.

In addition, all the experimental variables involved in the preparation of the immunocaptors and in the formation of sandwich immunocomplexes on the immunocaptors surface were optimized for the single determination of bovine, ovine and caprine IgGs, respectively. The ratio between the amperometric responses obtained in the presence of 250 ng mL-1 of the target IgG standard (S) and in its absence (N) was adopted as the selection criterion. The results obtained are displayed in Figures S1 (immunocaptors preparation) and S2 (sandwich-immunocomplexes formation) in the Supporting Information. The tested variables and ranges as well as the selected values are summarized in Table S2 (also in the Supporting Information). The volume of the commercial HOOC-MBs suspension31 and the variables involved in the amperometric transduction (applied potential and H2O2 and HQ concentrations29,32,33) were taken from previous works. It is worth highlighting from these optimization studies the high non-specific current (in the absence of target IgG) obtained when large concentrations of CAb (panels 1-2a in Figure S1) or HRP-DAb (panels 1-2b in Figure S2) specific for bovine and ovine IgG are used. It is also important to note that better S/N ratios were obtained for all IgGs when the capture by the immunocaptors and labeling with the specific

0

50

100

150

200

250

[IgG Standard], ng mL-1

Figure 3. Calibration plots constructed with the developed immunoplatforms for bovine 1), ovine 2) or caprine 3) IgG standards. Error bars were estimated as triple of the standard deviation of three replicates.

Table 1. Analytical and operational characteristics achieved for the amperometric determination of bovine (1), ovine (2) and caprine (3) IgG standards with the developed immunoplatforms. Bov-IgG (1)

Ovi-IgG (2)

Cap-IgG (3)

R2

0.9997

0.9968

0.9991

Slope, nA mL ng-1

6.1 ± 0.1

7.2 ± 0.5

3.5 ± 0.1

Intercept, nA

181 ± 13

196 ± 48

68 ± 12

L.R., ng mL-1

2.6  250

2.7  250

2.2  250

LOD, ng mL-1

0.74

0.82

0.66

4.5

4.6

3.2

67

67

67

RSD(n=8, -1 mL ), %

100

Stability, days


ng

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Analytical Chemistry

The relative standard deviation (RSD) values, calculated from the amperometric responses obtained with 8 different immunosensing platforms prepared in the same way for 100 ng mL-1 of the target IgG, were 4.5, 4.6 and 3.2 % for bovine, ovine or caprine IgG, respectively thus showing the robustness of the protocols used for the preparation of the MBs with the sandwich immunocomplexes and in the amperometric transduction at the SPCEs. The storage stability of the prepared magnetic immunocaptors was evaluated by storage at 4 ºC in 50 µL of filtered PBS. Each working day, the amperometric responses obtained with the immunoplatforms prepared from the stored immunocaptors in the absence (N) and in the presence (S) of 100 ng mL-1 of each target IgG, were measured. Results shown in Figure S3 (in the Supporting Information) demonstrate that no significant decrease in the S/N ratio was apparent during at least 67 days (no longer times were assayed). These data confirmed an excellent storage stability of the magnetic immunocaptors, which can be prepared and stored allowing the determination to be completed in just 30 minutes. These first immunosensing platforms reported in this paper for the determination of animal-species-specific IgGs provide better LOD values than those claimed for most of the available ELISA kits (summarized in Table S1 in the Supporting Information) in significantly shorter analysis time (30 vs. 60120 min). It should be remarked the significant improvement achieved in the LODs with respect to the ELISA kits marketed by ZEULAB which use the same immunoreagents (LODs of 0.74, 0.82 and 0.66 ng mL-1 vs. 70.0, 14.0 and 10.0 ng mL-1 for bovine, ovine and caprine IgGs, respectively) and in 3-times shorter assay time. The achieved improvement in sensitivity can be very advantageous in routine detection of low levels of adulteration in cheese, which requires a higher sensitivity due to matrix effects and loss of IgG into the whey and due to proteolysis and denaturation during cheese manufacture.3 Additional advantages of the developed immunoplatfoms include their compatibility with multiplexing and the use of affordable instrumentation for point-of-care determinations.

Selectivity of the immunosensing platforms. The selectivity of the immunoplatforms was tested both in buffered solutions supplemented with the corresponding IgG standards and in milk samples with different endogenous IgG contents. The results are displayed in Figure 4. Panels 13a show that the amperometric responses obtained with each immunoplatform were significantly different from the background current only in the presence of the target IgG. In addition, similar S/N ratios were obtained for the target IgG in the absence and in the presence of the non-target IgGs thus confirming the absence of cross-reactivity between different IgGs and the excellent selectivity of the used antibodies. Panels 13b in Figure 4 confirm the excellent selectivity also in milk samples. It is important to note that a clear discrimination is shown in milk from species that are extremely similar, such as cow and buffalo or sheep and goat, (panels b in Figure 3).6 Indeed, it has been widely reported that the discrimination between cows’ and buffalo’s milk is very challenging due to the high homology between milk proteins of these two species.8

-i, A 1.0

0.0 ng mL-1 Bov-IgG 100 ng mL-1 Bov-IgG

1 a) S/N 8

-i, A

1 b)

1.0

6 4

0.5

0.5

Pasteurized

2 0.0

-i, A

B.S.

Ovi-IgG

0.0 ng mL-1 Ovi-IgG 100 ng mL-1 Ovi-IgG

Cap-IgG

0

UHT

0.0

B

O

C

Buf

B

O

2 a) S/N -i, A 6 4

0.5

Pasteurized

1.0

0.5

2 0.0

-i, A 0.6

B.S.

Bov-IgG

0.0 ng mL-1 Cap-IgG 100 ng mL-1 Cap-IgG

Cap-IgG

0

UHT

0.0

B

O

C

Buf

B

O

3 a) S/N -i, A 8 6

0.4

B

2 b)

8

1.0

C

C

B

3 b)

0.9

Pasteurized

0.6

4 0.2 0.0

2 B.S.

Bov-IgG

Ovi-IgG

0

0.3 UHT

0.0

B

O

C

Buf

B

O

C

B

Figure 4. Selectivity of the developed bovine (1), ovine (2) or caprine (3) IgGs immunoplatforms for the determination of standards in buffered solutions (a) and in different milk samples (b). Comparison of the amperometric responses provided by the developed immunosensing platforms for the determination of 0.0 and 100.0 ng mL−1 bovine (1), ovine (2) or caprine (3) IgG standards in the absence (buffer solution, B.S.) or in the presence of 100.0 ng mL−1 non-target IgG standards a). Comparison of the amperometric responses in different diluted milk samples: raw (1/5,000), pasteurized (1/5,000) or UHT (1/100) from cow (B, blue bars), ovine (O, purple bars), caprine (C, green bars) or buffalo (Buf, yellow bars) b). Error bars were estimated as triple of the standard deviation of three replicates.

Applications to the analysis of milk samples and detection of milk adulterations. The bioplatforms were applied to the determination of the target IgGs in milk samples from different species (bovine, ovine and caprine) and with different treatments (raw, pasteurized and UHT). A 100-times or more dilution of the milk samples avoided matrix effects. In fact, the slope values of the calibration plots constructed in 100-time diluted cow, ovine and caprine milk were 5.8 ± 0.7, 7.8 ± 0.7 and 3.7 ± 0.3 nA mL ng-1, respectively, which are not significantly different than those obtained in calibration graphs prepared with bovine, ovine and caprine IgG standards (6.1 ± 0.1, 7.2 ± 0.5 and 3.5 ± 0.1 nA mL ng-1). Accordingly, quantification was accomplished by simple interpolation of the amperometric responses measured with the developed platforms into the calibration plots constructed with standard IgG solutions (Figure 3). The results obtained for the analyzed samples as well as the dilution used in the working protocol are summarized in Table 2. The calculated concentrations are, in general, in good agreement with the data reported by other authors as well as with the loss of IgG activity after the heat treatments applied to the milk samples. Pasteurized milk can retain 2575 % of the IgG concentration compared with raw milk, while no IgG



activity was detected after ultra-high temperature (UHT)treatment due to denaturation of the target epitope.18 As expected, larger concentrations of IgGs were found in colostrum samples. The higher variability of the results reported for goat colostrum can be attributed to the many factors affecting the IgG concentration in these samples including season of calving, lactation number, dry period length, intercalving interval, volume of colostrum produced, bacterial infections overcome by the animal, age, breed, etc..14 Table 2. Determination of IgGs (in mg mL-1) in different milk samples with the developed electrochemical immunoplatforms. Comparison with the reported data. Ani mal

Cow

Milk type (Dilution)

BovIgG (1)

Raw (1/5,000)

(0.52 ± 0.05)

ND

ND

Pasteuriz ed (1/5,000)

(0.050 ± 0.001)

ND

ND

UHT (1/100)

ND

ND

ND

ND16,34 0.003 ppm36

Colostru m (1/100,00 0)

ND

(6.2 ± 0.3)

ND

(9.25)37 (≥0.47)15

Raw (1/5,000)

ND

(0.64 ± 0.07)

ND

(0.25)37 (0.2)15

Pasteuriz ed (1/5,000)

ND

(0.53 ± 0.03) (17.5 % IgG activit y lost compa red to raw milk)

ND

10±30 % of the IgG activity is lost after HTST pasteurisation (72 °C/15 s)16

UHT (1/100)

ND

ND

ND



Shee p

Colostru m (1/100,00 0) Goat

ND

Raw (1/5,000)

ND

Pasteuriz ed

ND

OviIgG (2)

CapIgG (3)

Values reported in the literature (0.030.71)34

ND

(26 ± 4)

ND

(1.1 ± 0.2)

ND

(0.86 ± 0.04)

(0.71)16 0.5935 (0.040.24)34 (0.50.7)16

32.98 14.3938

or

Page 6 of 9 (1/5,000)

UHT (1/100)

ND

ND

(24.5 % IgG activit y lost compa red to raw milk)

lost after HTST pasteurisation (72 °C/15 s)16

ND



ND: non-detectable; HTST: high temperature/short time.

The possibility of detecting milk adulteration with the developed immunoplaftoms was tested considering the most common adulterations, i.e. sheep, goat or buffalo milk adulteration with cow milk (Figure 5a), cow or goat milk adulteration with sheep milk (Figure 5b) and sheep milk adulteration with goat milk (Figure 5c). To perform these studies raw milk samples were adulterated with 0.1 and 0.5 % of the corresponding animal milk and after diluting the samples 100-times, the amperometric responses of the immunosensor platform developed for bovine IgGs (samples adulterated with cow milk), ovine IgGs (samples adulterated with sheep milk) and caprine IgGs (samples adulterated with goat milk) were measured. The results displayed in Figure 5 show fairly well the ability of the immunoplatforms to detect unequivocally the most common types of adulteration with milk from other species at least at levels of 0.1 %. It is important to note that this level is 10-times lower than the current EU permitted level of cows’ milk, 1.0 % (Commission Regulation (EC) No 273/2008)3,8 and similar to those achieved by the ELISA kits commercialized by ZEULAB that use the same immunoreagents. However, the significantly lower LODs obtained for IgG standards and the fact that 100-fold diluted samples were analyzed will open the way to detect adulterations at lower levels (by using less diluted samples), thus making the developed immunoplatforms even more competitive against ELISA methodologies.

±

506035 6539 47.913 (0.10.4)35 (3)39 (≤12)13 10±30 % of the IgG activity is

Figure 5. Amperometric responses measured with the developed immunoplatforms for bovine a), ovine b) and caprine c) IgGs to detect adulteration in 100-times diluted raw milks indicated on the


Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry x-axis unadulterated or adulterated with 0.1 and 0.5 % cow a), sheep b) and goat c) milk. Dashed line: blank response provided by the biosensors in the absence of any IgG. Error bars were estimated as triple of the standard deviation of three replicates.

Immunosensing platforms for the detection of total IgGs. Due to the problems caused in the dairy industry by adulteration with colostrum,13,14 we implemented a simple methodology to detect this particular adulteration and ensure milk free from colostrum. The methodology relies on the detection of the three target IgGs by using a mixture of target specific modified MBs and amperometric detection at a single SPCE. The modified MBs were commingled together and incubated in the sample supplemented with the three HRP-DAbs, and then captured on the surface of a SPCE. Figure 6 demonstrates that this methodology allows a clear discrimination between milk samples unadulterated and adulterated with sheep colostrum between 5 and 100 %. It is also possible the detection of 5 % ovine colostrum with a smaller IgG concentration in raw ovine milk samples (look at the purple bars on the right side of the Figure 6). Therefore, the developed strategy can be used as an attractive, effective and rapid tool (30 min) for the screening of milk regarding adulteration with colostrum.

b), 1.0 % raw caprine milk c), and 20 % ovine colostrum. The obtained results, which are exemplified for ovine milk in Figure 7, demonstrate the usefulness of the multiplexing platforms to detect adulteration of milk with milk from other animal species (Figures 6b and c) or colostrum (Figure 6d).

Figure 7. Multiplexing platform for the detection of bovine, ovine, caprine and total IgGs in milk samples at SPC4Es. Amperometric traces (left) and measured currents (right) obtained in the analysis of 100- (W1W3) or 10,000-times (W4) diluted raw sheep milk: unadulterated a) or adulterated with 1.0 % raw bovine milk b), 1.0 % raw caprine milk c), and 20 % ovine colostrum. For comparative purposes, amperometric traces recorded in the absence of bovine, ovine and caprine IgGs (buffered solutions) are displayed as black striped lines. Error bars were estimated as triple of the standard deviation of three replicates.

CONCLUSIONS

Figure 6. Schematic display of the methodology involved for total IgGs determination at a SPCE a). Amperometric responses obtained in the absence and in the presence of 1 mg mL-1 of bovine, ovine and caprine IgGs, and for a mixture of the 3 IgGs (total concentration 1 mg mL-1), in raw milk samples, and in raw ovine milk spiked with ovine colostrum at different percentages b). All IgG standards and milk samples were 10,000 times diluted. Error bars were estimated as triple of the standard deviation of three replicates.

Multiplexing platform for the detection of bovine, ovine, caprine and total IgG. A methodology allowing the identification of the animal origin of a milk sample as well as possible adulteration events with other animal milks or colostrum in a single run was developed. To do that, we performed the multiplexed detection of bovine, ovine, caprine and total IgGs at SPC4Es. Batches of MBs designed for the single detection of bovine, ovine and caprine IgGs or for total IgGs were prepared and magnetically captured on each of the 4 working electrodes (Figure 7). This Figure also shows actual amperometric traces recorded with the multiplexing platform for the analysis of a 100- (W1W3) or 10,000-times (W4) diluted raw ovine milk sample, both unadulterated a) and adulterated with 1.0 % raw bovine milk

This work reports the first electrochemical biosensing platforms suitable to detect milk adulteration processes with milk from other animal species or colostrum in just 30 minutes. The designed bioplatforms involved the implementation of sandwich-type immunoassays for the sensitive and selective determination of bovine, ovine and caprine IgG on magnetic microparticles. The extent of immune-recognition events was individually or multiplexed monitored by amperometry at disposable electrodes using the H2O2/HQ system. The developed platforms allow the determination of the target IgGs standards with LODs of 0.74, 0.82 and 0.66 ng mL-1 for bovine, ovine and caprine IgG, respectively. The methodology is able to detect low levels (0.1 %, v/v) of adulteration in both crude and heat-treated milk. In addition, the versatility of the bioplatforms allows the screening of milk adulterated with colostrum and multiplexed measurements able to provide both the total and the individual levels of each target IgG. The multiplexing platforms can give relevant information on the sample analyzed, such as milk speciation, commercial processing (thermal treatment) undergone and whether it has been adulterated with milk from other animal species or with colostrum. These smart biosensing platforms can be advantageously compared with conventional ELISA methodologies in terms of simplicity, cost, assay time, feasibility for multiplexing and portability of the required instrumentation. These features make the bioplatforms particularly attractive analytical tools for routine and/or on site milk inspection against species substitution or admixture, thus guaranteeing that milk and



dairy products are unadulterated and accurately labeled and protecting both producers and consumers from this serious fraud. The simplicity of the developed methodologies makes them easy to use by little training staff during milk collection at the farm or upon arrival at dairies.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details of bovine, ovine and caprine IgGs immunocaptors, Single or multiplexed detection of individual bovine, ovine, caprine or total IgGs, Amperometric measurements, Analysis of milk samples, Tables S1-S2 and Figures S1-S3 (PDF).

AUTHOR INFORMATION Corresponding Author *Tel.: +34 686702631. E-mail: [email protected] (Sara Benedé).

*Tel.: +34 913944219. Fax: +34 913944329. [email protected] (Susana Campuzano). *Tel.: +34 913944315. Fax: +34 913944329. [email protected] (José M. Pingarrón).

E-mail: E-mail.

Author Contributions VRVM and EP performed research. VRVM, EP, SB, MG, AJR, SC and JMP designed the experiments. VRVM, EP, SC and JMP wrote the manuscript. LM and PGM provided the immunoreagents. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The financial support of the Spanish Ministerio de Economía y Competitividad, CTQ2015-64402-C2-1-R Research Project and the TRANSNANOAVANSENS Program from the Comunidad de Madrid (P2018/NMT-4349) and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (E. Povedano) and Universidad Complutense de Madrid (V. Ruiz-Valdepeñas Montiel) are also gratefully acknowledged. S. Benedé acknowledges the financial support from MINECO through the “Juan de la Cierva” program. The authors would like to acknowledge also Zeulab S.L. Company for kindly providing the immunoreagents used.

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