Patent Application
20190118174
The present invention refers to the field of microfluidics, in particular it shows that microfluidic chips are especially suitable for use in a number of immunoassays (such as ELISA immunoassays) for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
There is an increase of the market of biosensing in the agro-food sector, where the ultrasensitive and low cost detection of food contaminants is required.
In particular, the companies in the poultry industry perform routine control tests for the presence/absence of pathogens such as Salmonella, E. coli or Campylobacter wherein the detection protocol is broadly regulated in the meat itself as well as in boot swabs, work boots, work tables, poultry fattening, laying hen farms, etc.
For the specific detection of Salmonella, different methods have been developed, based on an immunoassay such as ELISA, or on other suitable assays such as PCR and stock culture, to reduce the time required for the detection of this pathogen, because standard culture methods, such as the International Organization for Standardization Method 6579 (ISO) and the United States Food and Drug Administration's Bacteriological Analytical Manual Chapter 5: Salmonella (FDA), although they have a very low detection limit of 9 CFUs/mL (colony forming units per mL) for both poultry meat and poultry meat products, require up to 5 days (including biochemical and serological confirmations; ISO, 2002; FDA, 2007) to finalize the methods, and are thus not efficient in the routine monitoring of large numbers of samples. In this context, rapid, accurate, and economical methods, are crucial both for the industry and for laboratories reporting results to governmental authorities for taking legal actions. One of these methods is the Vitek immunodiagnostic assay (VIDAS; Biomérieux, Marcy L'Etoile, France), an automated enzyme-linked fluorescent assay-based system that allows for the accurate and rapid screening of large numbers of samples for the presence of Salmonella by the Vitek immunodiagnostic assay system Salmonella (VIDAS SLM) method. The detection limit of VIDAS ESLM for both poultry meat and poultry meat products, was determined to be 90 cfu/mL, in 48 hours.
However, to date all known tests, including Vitek immunodiagnostic assay, for screening samples for the presence of Salmonella require qualified staff and specific laboratory equipment, significantly delaying the provision of the results. If we take into account the fact that Salmonella-positive result in any of the known tests may imply the slaughter of all chickens in a housing unit, unless it can be treated early, it is a major issue for the food industry to identify the presence of pathogens as quickly and efficiently as possible in order to take the appropriate measures.
The present invention provides a rapid, highly sensitive and specific method for the identification of a wide variety of analytes, including pathogens such as Salmonella, E. coli or Campylobacter, in an efficient manner.
Disclosed herein, in various exemplary embodiments, we show that microfluidic chips are especially suitable for use in a number of immunoassays for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
These devices are useful for detecting the presence of one or more target analytes in one or more sample fluids. Methods and processes of making and using such devices are also disclosed in the examples.
Therefore, in particular the present invention refers to the in vitro use of a microfluidic kit or device comprising a support or substrate, wherein said support or substrate comprises at least one channel in the substrate, the channel comprising an inlet, an outlet, and a flow-path connecting the inlet and outlet, wherein the inlet and outlet together define a midplane, and a portion of the flowpath travels transversely across the midplane, wherein the portion of the flowpath that travels transversely across the midplane includes a recognition site or sensing area for detecting a target analyte;
for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
It is noted that midplane is a plane passing through the channel in such a way as to divide it into symmetrical halves and sensing area is defined as the portion of the metal-chetale activated surface functionalized with the antibody, identified inside the flowpath that travels transversely across the midplane between the inlet and outlet.
For the purpose of the present invention, the following definitions are included below:
The present invention provides a solution for offering a highly specific and sensitive method for the identification of a large variety of analytes, such as food pathogens as Salmonella, E. coli or Campylobacter, allergens such as Ara H 1 or other analytes such as collagen or albumin, in a rapid an efficient way.
For this purpose, the authors of the present invention combined the use of a number of functionalized surfaces with antibodies capable of detecting the target analyte by using the Heatsens technology (see definitions). Therefore, controlled heat generation in combination with functionalized surfaces with antibodies was chosen as the basis for the new generation of detection systems developed in the present invention. The phases of the protocol for the detection of the analyte used herein are summarized in the flowchart shown in
In order to implement this technology, we first tested the adequate coupling of antibodies specific against Salmonella typhymurium using the ELISA technique and a dot-blot assay format. As analyte, an attenuated Salmonella typhymurium from BacTRace (https://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=415&Product_ID=952) was used as model to implement and optimize the assay.
The detection of Salmonella using this standard methodology (ELISA) using enzymes as labels of the analyte presence, achieved a limit of detection (LOD) of 1.400 CFUs in the case of the ELISA assay and 3.125 CFU in the case of the detection with a dot-blot assay format, as shown in
The above-mentioned surfaces were selected due to their different capacities for their functionalization with antibodies and for their thermal conductivity, reported herein below:
As shown through-out the present invention, an ideal surface to be used as the detection surface has to: i) allow the use of functionalization methodologies to ensure an oriented binding, and ii) have a high thermal conductivity. Increasing the thermal conductivity of the detection support used for HEATSENS will improved the sensitivity of the immunodetection of the analyte, since the heat released by the metal nanoparticles interacting with the analyte, will be measured in a faster and more precise way from the thermal detector.
We herein below describe the functionalization of the different surfaces tested herein:
To perform the incubation with the analyte of the nitrocellulose or PVDF membranes and the patterned TiO2 film, they were incubated with 200 μl of the different concentrations of the analyte in a buffer (respectively buffer phosphate, peptone culture media, and real sample) for 30 min at 37° C. with agitation. The incubated supports were washed two times adding 400 μl of washing solution (PBS buffer with 0.5% of BSA and 0.5% of Tween), and were incubated at room temperature for 5 minutes with agitation. When this washing step was finished two additional washing steps were performed adding 400 μl of sodium phosphate buffer 10 mM pH 7, incubating the surfaces at room temperature for 5 minutes with agitation. The final step of the detection was the incubation of the support with 20 μg/ml of streptavidine@nanoprisms, diluted in blocking buffer (PBS buffer with 5% of BSA and 0.5% of Tween) for 30 min at 37 C. The surfaces were then dried for 15 minutes at 37° C.
For each experiment we validated the specific interaction of the antibodies with the salmonella, introducing the following controls:
The detection of Salmonella was first made in a semi-quantitative way using a thermal paper coupled to the functionalized membrane/support and displayed as the burning of the thermal paper. The support used was PVDF functionalized with capture antibody for testing the capture and of course detection, of the different dilutions of salmonella, in a range between 150 CFUs and 6.000 CFUs in 200 microliter samples. The illumination of the membrane, after incubation with the nanoprisms functionalized with the detection antibody, achieved the visual detection of 150 CFUs in a 200 microliter sample of Salmonella, detection shown in
However, the above visual method did not achieve a satisfactory detection limit for use in food contaminated samples wherein the pathogen is scarcely present in just a few CFUs/ml such as in an amount <90 CFUs/ml.
In order to solve this problem, the authors of the present invention try to use a quantitative detection using commercial thermopiles. In this sense, it is noted that the heat released by nanoprisms upon IR illumination can be measured by using an IR thermopile, such as a MIX90620 from Melexis. This thermopile is suitable to detect thermal radiation and measure temperatures without making contact with the sample.
The MIX90620 thermopile contains 64 IR pixels with dedicated low noise chopper stabilized amplifier and fast ADC integrated. A PTAT (Proportional to Absolute Temperature) sensor is integrated to measure the ambient temperature of the chip. It requires a single 3V supply (+0.6V) although the device is calibrated and performs best at VDD=2.6V. The MLX90620 is factory calibrated in wide temperature ranges: −40 . . . 85° C. for the ambient temperature sensor −50 . . . 300° C. for the sample temperature. Each pixel of the array measures the average temperature of all objects in its own Field Of View (called nFOV).
For the quantitative detection, salmonella was directly immobilized onto a PVDF support at different CFUs dilutions, in the range within 375 to 6.000 CFUs, in 200 microliter samples, in particular a dilution containing 375 CFUs and a dilution containing 700 CFUs were used. Detection was performed in a quantitative way by measuring the increment of temperature generated by the presence of nanoprisms interacting with the analyte, as shown in
As a negative control, we measured the temperature increased of those membranes that followed the same protocol of detection but were not incubated with salmonella. As expected, in the absence of salmonella, the nanoprisms did not interact with the membrane, as we did not observe an increase of the increment of temperature of this control.
In the presence of the salmonella, previously diluted in buffer phosphate and directly adsorbed onto surface, the increment of temperature of 375 CFU was of approx. 19° C., meanwhile 700 CFU of salmonella, generated an increment of approx. 27° C. However, as with the visual method, we did not achieve a satisfactory detection limit for use in food contaminated samples wherein the pathogen is scarcely present in just a few CFUs/ml such as in an amount <90 CFUs/ml.
To solve this problem, we then tried using supports other than PVDF and nitrocellulose, such as TiO2 patterned supports. Yet, as with the visual detection method and the quantitative detection methods shown so far, a satisfactory detection limit was again not achieved in a reliable way.
In order to solve this problem, we then tried combining the microfluidic technology with the Heatsens technology in order to carry out a series of immunoassays capable of detecting a target analyte with a satisfactory detection limit in a reliable way. For this purpose, the unmodified fabricated microfluidic chip illustrated in the materials and methods of the examples was used for testing the direct immobilization of two dilutions of salmonella. For this purpose, 10 μl of 60000 CFU/ml and 20000 CFU/ml (600 and 200 CFU in total on the surface, respectively) of Salmonella T. were adsorbed on the detection surface. After the direct immobilization of the pathogen, the surface was blocked with BSA and allow to react with biotinylated detection antibodies. Finally, they were washed and further reacted with streptavidin-AuNanoprisms solution.
In
In view of these results, we then used 10 μl of different concentrations (CFU/ml) of salmonella T, in a range between 0 and 240000 CFU/ml. These were directly adsorbed onto the microfluidic chip and detected with biotinylated antibodies anti-salmonella to measure the increment of temperature due to the presence of different concentrations of salmonella. Then the strepavidine@AuNprism interacted with the antibodies and every single sensing area was irradiated with an IR laser. The temperature of each chamber was measured, and the increment of temperature calculated.
The increase of temperature measured was due to the increased amount of CFUs directly adsorbed onto the surface of microfluidic chip.
Once shown that the microfluidic chip was suitable to be applied to the HEATSENS technology, we performed a sandwich type immunoassay for the detection of the selected pathogen by using a microfluidic chip. For this purpose, each micro-chamber of the microchip was functionalized with capture antibodies anti-salmonella by direct adsorption of (5 μL) 5 μg/ml of capture antibodies anti-salmonella onto the surface. Then, the salmonella's capture event was carried out in fluidic mode, as well as the detection and the interaction with the streptavidin-AuNprism, injecting 1 ml of sample, in each channel. The assay was carried out with 2 different concentrations of salmonella's CFU/ml, 200000 CFU/ml and 240000 CFU/ml diluted in buffer phosphate, respectively.
Once shown the effectiveness of an immunoassay in a sandwich format, we tried to improve the limit of detection of salmonella t., by decreasing the concentration of the pathogen in doped buffer. In this sense, 1500 CFU/ml of salmonella T. in PBS 1× was the first lower concentration detected in the first trial. For this purpose, a 1 ml sample was injected in the channel with a flow of 200 μl/ml. After injecting the sample, the channel was washed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/min for 4 min. Then the detection antibodies were allow to interact with its antigen using a 200 μl/ml for 2 minutes. The channel was rinsed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/min for 4 min. The streptavidin@AuNPr were injected into the channel. The flow was 200 μl/ml for 2 minutes. The channel was rinsed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/mim for 4 min and dried.
The same experiment was carried out using a real food sample, 25 g of chicken meat in 225 ml of peptone pre-enrichment culture media, doped with salmonella at different CFUs. The capture antibodies were adsorbed onto the microfluidic chip, and the surface blocked with 5% BSA in PBS1×-01% Tween, using a flow rate of 15001/min.
Then, the washing was carried out using a flow rate of 250 μl/min, by using a washing buffer.
The capture of salmonella in a 1 ml of real sample, as well as the detection with biotinylated detection antibodies, and the interaction with streptavidin@nanoprisms was performed by using a flowing at a flow rate of 15 μl/min.
The results of the immune analysis carried out in the microfluidic chip are shown in
The higher increment of temperature of the samples doped with salmonella, clearly indicates that HEATSENS in combination with the microfluidic technology is suitable for the ultrasensitive detection of few CFUs of bacteria in complex matrices such as the 25 g of chicken meat in 225 ml of peptone.
The increment of temperature due to the presence of salmonella in a real sample is slightly different from the one in buffer phosphate, because of presence of high amount of meat proteins which affect the specific interaction of the bacteria with the antibodies.
Once shown that the combination of HEATSENS with the microfluidic technology is suitable for the ultrasensitive detection of few CFUs of an analyte, we tried to improve this technology by modifying the microfluidic chip surface with carboxylic end groups used to immobilize covalently capture antibodies by the formation of stable amide bonds with their primary amines via EDC/sulfo-NHS reaction.
In this sense, the surface of each micro-chamber, previously activated with 10 mM EDC and 20 mM sulfo-NHS, was functionalized with 20 μl of 5 μg/ml of capture antibodies. After the covalent immobilization of the capture antibodies and the blocking of surface with BSA 5% in PBS1×/0.1% Tween for 1 hour at 37 C, the chip was connected to the peristaltic pump and washed with washing buffer using a flow rate of 300 μl/min for 4 minutes. 1 ml of 30 CFU/ml of Salmonella T., were allow to flow inside the microfluidic channel for 1 minute at a flow rate of 150 μl/min, then the channel was washed with a buffer solution using a flow rate of 300 μl/min for 4 minutes. 400 μl of biotinylated detection antibodies were then flowed inside the channel.
The results depicted in
We then tried an oriented immobilization of the antibodies through metal-chelation. Immobilization was accomplished through the metal-chelation to histidine-rich metal binding site in the heavy chain (Fc) of the antibody or to poly-His-tag sequence fused in proteins. Since the metal binding site is either in the C- or N-terminus, antibodies and His-tagged proteins bound in this fashion to the surface are oriented with the combining site directed away from the surface thus allowing maximal antigen binding or a favourable protein orientation. Furthermore, oriented immobilization through metal-chelation also results in a stable antibody immobilization since binding constants for metal-chelation immobilization are very high due to the combination of the chelate effect of histidine binding, and target binding of multiple metal-chelate groups. Dissociation constants are estimated to be between 10−7 to 10−13 M−1. For many applications, this provides binding strengths comparable to antigen-antibody interaction. On the other side, experimental conditions of antibody attachment for oriented immobilization of antibodies through metal-chelation are milder than those employed for covalent oriented immobilization procedure. As an advantage, the antibody binding to the chelate could be also modulated as convenience to be reversible or irreversible. In addition, it is also more versatile since it can be also employed for immobilization of his-tagged recombinant proteins.
In order to achieved an oriented immobilization of the capture antibodies, the microfluidic chamber chips were functionalized with metal-chelate complexes in a stepwise modification of their surface. Firstly, the surfaces were functionalized with aryl amine compounds containing carboxylic groups such as for example 3-(4-Aminophenyl)propionic acid, 3-Aminophenylacetic acid, 4-Aminophenylacetic acid or 4-(4-Nitrophenyl)butyric acid. For this specific example we used PhBut, even though for the immobilization of different biomolecules, it would be more appropriate the use of aryl amine compounds carrying different lengths of n-alkyl carboxylic acids in a range between 2 and 16 carbons.
Carboxylic groups introduced by covalent grafting of the aryl radical of diazotated PhBut (Scheme II) were activated by esterification with SNHS catalyzed by EDC to facilitate the covalent linkage of the ANTA-M(II) (Cu2+, Ni2+, Co2+) complex (Scheme III) through the free amino groups. Then, they were incubated with 20 μl of 5 μg/ml of capture antibodies. The resulting NTA-M(II) complex termination contains two free coordination sites occupied by water molecules to be replaced by histidine residues of capture antibodies giving rise to their oriented immobilization. Later, the chip was connected to the peristaltic pump and washed with washing buffer using a flow rate of 300 μl/min for 4 minutes. 1 ml of 30 CFU/ml of salmonella T., was allow to flow inside the microfluidic channel for 1 minute at a flow rate of 150 μl/min, then the channel was washed with buffer using a flow rate of 300 μl/min for 4 minutes. 400 μl of biotinylated detection antibodies was then flowed inside the channel.
Interestingly, the temperature increment due to the presence of Salmonella for this type of immobilization was higher than those obtained for the respective controls and even higher than those obtained in previous results for direct adsorption and covalent immobilization. In this sense, a comparative study between the different immobilization methods was carried out. The comparison of the different strategies of antibody surface functionalization is displayed in
The advantageous antibody oriented immobilization shown in example 6, was then tested for the detection of salmonella in a real sample. The result is reported in
The temperature increment, due to the presence of salmonella in the real sample, on an oriented antibody immobilized microfluidic chip surface, was also higher than those obtained for the respective controls. After building the calibration curve, the measurement of the increment of temperature due to the known different concentrations of salmonella and to the unknown concentration of pathogen in the real sample was determined, as reported in the
Therefore, the microfluidic technology was thus selected as the best approach for the fabrication of a preferably disposable cartridge required to perform a HEATSENS protocol for analyte detection. Moreover, the microfluidic technology in combination with an oriented configuration of the capture biomolecules (such as antibodies) has been shown herein as an excellent approach for the fabrication of a preferably disposable cartridge required to perform a HEATSENS protocol for analyte detection.
Lastly, it is important to note that as clearly illustrated in examples 8, 9 and 10, the combination of the microfluidic technology and the Heatsens technology is suitable for the detection and quantification of a large variety of analytes such as, but not limited to, microorganisms, additives, drugs, pathogenic microorganisms such as a food pathogens, food components, environmental contaminants, pesticides, nucleotides, biomarkers such as medical biomarkers or toxic compounds etc. Therefore, the sensor systems described herein are not limited to any specific analyte.
Use of the Microfluidic Technology in Combination with the Heatsense Technology
Disclosed herein, in various exemplary embodiments, we show that microfluidic chips are especially suitable for use in a number of immunoassays for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
These devices are useful for detecting the presence of one or more target analytes in one or more sample fluids. Methods and processes of making and using such devices are also disclosed in the examples.
Therefore, a first aspect of the invention refers to the in vitro use of a microfluidic kit or device comprising a support or substrate, wherein said support or substrate comprises at least one channel in the substrate, the channel comprising an inlet, an outlet, and a flow-path connecting the inlet and outlet, wherein the inlet and outlet together define a midplane; and a portion of the flowpath travels transversely across the midplane, wherein the portion of the flowpath that travels transversely across the midplane includes a recognition site or sensing area for detecting a target analyte;
for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
In a preferred embodiment of the first aspect of the invention, the portion of the flowpath travels transversely across the midplane multiple times. In another preferred embodiment of the first aspect of the invention, the portion of the flowpath may travel substantially perpendicularly across the midplane. In another preferred embodiment of the first aspect of the invention, the portion of the flowpath may travel continuously towards the outlet from the inlet. In another preferred embodiment of the first aspect of the invention, the device has a plurality of channels. In another preferred embodiment of the first aspect of the invention, the device has a plurality of micro-chambers with recognition sites in each one or more channels. In yet another preferred embodiment of the first aspect of the invention, the inlet of each channel is connected to a common loading channel. In still another preferred embodiment of the first aspect of the invention, the device comprises the characteristics of the microchip or device described in the materials and methods section of the examples.
In addition, it is noted that the substrate or surface of the device of the first aspect of the invention, may be made from a variety of materials such as thermoplastic materials, silicon, metals, or carbon. Preferably, it may be made by poly(methyl methacrylate), polystyrene, poly(dimethylsiloxane), polyethylene terephthalate, polyethylene, polypropylene, polylactic acid, poly(D,L-lactide-co-glycolide), polycarbonate, cyclic olefin copolymers, silicon, glass etc.
As illustrated in the examples (see examples 6 to 9), functioanalizing the sensing area of the microchip's surface improves the characteristics of the sensor by providing a covalent or oriented configuration of the capture biomolecules.
Thus, in another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the portion of the flowpath that travels transversely across the midplane that includes a recognition site or sensing area is functionalized with one or more carboxylic functional groups or epoxy functional groups or amine functional groups or thiol functional groups or azide functional groups or halides or maleimide functional groups or hydrazyde functional groups or aldehydes or alkynes groups.
Different manners of functionalizing these types of surfaces with the above mentioned functional groups are shown in the examples. Anyhow, in general, if the support or substrate is made of:
Preferably, any of the above surfaces is functionalized with carboxylic functional groups. More preferably, the support or the microfluidic chip or device is made of a thermoplastic material and the diazonium aryl compound is represented by formula I or II below:
wherein R is an alkyl group having from 1 to 15 carbon atoms or an ethylene group; and Z is a carboxylic group, an epoxy group, an amine group, a thiol group, an azide group, a halide, a maleimide functional group, a hydrazyde functional group, an aldehyde group or an alkyne group, preferably a carboxylic or epoxy group; or
wherein R is an alkyl group having from 1 to 15 carbon atoms.
Preferably the diazonium component of formula I or II above is place or sited in the para or meta position with respect to the alkyl or ethylene component of any of these formulae. Examples of aryl amine compounds suitable for producing the diazonium aryl compound of any of formula I or II above are: 3-(4-Aminophenyl)propionic acid, 3-Aminophenylacetic acid, 4-Aminophenylacetic acid, 4-(4-Nitrophenyl)butyric acid or 4-(4-Aminophenyl)butyric acid (see examples).
In a further preferred embodiment of the first aspect of the invention, the surface of the microchip or device is further modified or functionalized with a chelating agent preferably selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt, nitrilotriacetic acid (NTA) metal (II) salt, iminodiacetic acid (IDA) metal (II) salt, Ethylenediaminetetraacetic acid (EDTA) metal (II) salt, diethylenetriaminepentaacetic acid (DTPA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+. This is accomplished by direct reaction of the chelating agent with any of the activated functional groups referred to above (with the exception of those groups like the epoxy groups that do not need to be activated to directly react with the chelating agent), wherein:
Preferably, the support is made of a thermoplastic material and the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC).
More preferably, the support is made of a thermoplastic material, the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and the chelating agent is Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+.
As illustrated in the examples, activation of the microchip's surface with a chelating agent such as the ANTA metal (II) salt is especially advantageous to achieve an oriented configuration of the antibody resulting in an improved sensing platform.
In a further preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the portion of the flowpath that travels transversely across the midplane that includes a recognition site or sensing area may comprise:
Preferably, said recognition molecule can be selected from, but not limited to, the list consisting of: peptides, polysaccharides, toxins, protein receptors, lectins, enzymes, antibodies, antibody fragments, recombinant antibodies, antibody dendrimer complexes, nucleic acids, (DNA, RNAs), peptide nucleic acids (PNAs), molecular imprints. Preferably, said recognition molecule is an antibody, a fragment thereof or a recombinant antibody.
In a second aspect of the invention, the kit or device of the first aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In a third aspect of the invention, the kit or device of the first aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In a fourth aspect of the invention, the kit or device of the first aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
Preferably the kit or a device of any of the second to fourth aspects of the invention, further comprises a device capable of detecting the heat generated by the metal nanoparticles when they are irradiated with the external light source selected from the list consisting of infrared cameras or thermopiles.
A fifth aspect of the invention refers to the use of the device according to any of the precedent aspects of the invention, wherein the analyte is a microorganism, additive, drug, a pathogenic microorganism such as a food pathogen, a food component, an environmental contaminant, a pesticide, a nucleotide, a biomarker such as a medical biomarker or a toxic compound. Preferably, the target analyte is selected from the list consisting of Salmonella, Campylobacter, collagen, albumin and Ara H1.
Microfluidic Device or Chip Having a Sensing Area Functionalized for an Antibody Oriented Immobilization.
As established in examples 6 to 9 by functionalizing the sensing area of a microchip or device so that a capture biomolecule such as an antibody has an oriented configuration provides clear advantages for the detection of an analyte in a sensor system combining the microchip technology with the Heatsens technology.
Thus, a sixth aspect of the invention refers to a kit or device comprising a support or substrate, wherein said support or substrate comprises at least one channel in the substrate, the channel comprising an inlet, an outlet, and a flow-path connecting the inlet and outlet, wherein the inlet and outlet together define a midplane; and a portion of the flowpath travels transversely across the midplane, wherein the portion of the flowpath that travels transversely across the midplane includes a recognition site or sensing area for detecting a target analyte;
wherein the portion of the flowpath that travels transversely across the midplane that includes a recognition site or sensing area is functionalized with a chelating agent.
In a preferred embodiment of the sixth aspect of the invention, the chelating agent is preferably selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt, nitrilotriacetic acid (NTA) metal (II) salt, iminodiacetic acid (IDA) metal (II) salt, Ethylenediaminetetraacetic acid (EDTA) metal (II) salt, diethylenetriaminepentaacetic acid (DTPA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+, preferably Cu2+. Preferably the chelating agent is selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt or nitrilotriacetic acid (NTA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+, preferably Cu2+. This is accomplished by direct reaction of the chelating agent with an activated functional group, wherein:
Preferably, the support is made of a thermoplastic material and the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC).
More preferably, the support is made of a thermoplastic material, the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and the chelating agent is selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt or nitrilotriacetic acid (NTA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+, preferably Cu2+.
In a further preferred embodiment of the sixth aspect of the invention or of any of its preferred embodiments, the portion of the flowpath that travels transversely across the midplane that includes a recognition site or sensing area may comprise:
Preferably, said recognition molecule can be selected from, but not limited to, the list consisting of: peptides, polysaccharides, toxins, protein receptors, lectins, enzymes, antibodies, antibody fragments, recombinant antibodies, antibody dendrimer complexes, nucleic acids, (DNA, RNAs), peptide nucleic acids (PNAs), molecular imprints. Preferably, said recognition molecule is an antibody, a fragment thereof or a recombinant antibody.
In a seventh aspect of the invention, the kit or device of the sixth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In an eighth aspect of the invention, the kit or device of the sixth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In a ninth aspect of the invention, the kit or device of the sixth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
Preferably the kit or a device of any of the seventh to eighth aspects of the invention, further comprises a device capable of detecting the heat generated by the metal nanoparticles when they are irradiated with the external light source selected from the list consisting of infrared cameras or thermopiles.
Sensor System Combining the Microchip Technology with the Heatsens Technology Suitable for Detecting the Presence of an Analyte in a Sample Fluid
Additional aspects of the present invention refer to a full sensor system which combines the microchip technology with the Heatsens technology.
Therefore, a tenth aspect of the invention refers to a device or system for detecting the presence of an analyte in a sample fluid, comprising:
An eleventh aspect of the invention refers to a device or system for detecting the presence of an analyte in a sample fluid, comprising:
A twelfth aspect of the invention refers to a device or system for detecting the presence of an analyte in a sample fluid, comprising:
It is noted that the sensing area of the system or device of any of the tenth to twelfth aspects of the invention can be functionalized according to any of the techniques and with any of the functional groups described in the section entitled “USE OF THE MICROFLUIDIC TECHNOLOGY IN COMBINATION WITH THE HEATSENSE TECHNOLOGY”.
Preferably, the functionalization used allows an oriented configuration of the recognition molecule, preferably of an antibody.
In addition, it is further noted that the microchip or device mentioned as one of the components of the full sensor system of any of the tenth to twelfth aspects of the invention, may be further characterized as described in any of the embodiments described in the section entitled “USE OF THE MICROFLUIDIC TECHNOLOGY IN COMBINATION WITH THE HEATSENSE TECHNOLOGY”.
Processes for Functionalizing the Sensing Area of a Microchip or Device Suitable for Carrying Out Immunoassays by Detecting an Analyte by Using the Heatsens Technology.
As illustrated in the examples (see examples 6 to 9), functioanalizing the sensing area of the microchip's surface improves the characteristics of the sensor by providing a covalent or oriented configuration of the capture biomolecule.
As already established in the section entitled “USE OF THE MICROFLUIDIC TECHNOLOGY IN COMBINATION WITH THE HEATSENSE TECHNOLOGY” or in the section entitled “MICROFLUIDIC DEVICE OR CHIP HAVING A SENSING AREA FUNCTIONALIZED FOR AN ANTIBODY ORIENTED IMMOBILIZATION”, the sensing area of a microchip or device for use in carrying out immunoassays by detecting an analyte by using the Heatsens technology, can be functionalized in a number of different ways. The different ways of functionalizing the microchip or device depend on the type of material to functionalize and on the type of organic functional groups (such as carboxylic functional groups or epoxy functional groups or amine functional groups or thiol functional groups or azide functional groups or halides or maleimide functional groups or hydrazyde functional groups or aldehydes or alkynes groups) with which we wish to functionalize the sensing area of any of the microchips or devices shown through-out the present invention.
In this sense, it is noted that the substrate or surface of the microchip or device may be made from a variety of materials such as thermoplastic materials, silicon, metals, or carbon. Preferably, it may be made by poly(methyl methacrylate), polystyrene, poly(dimethylsiloxane), polyethylene terephthalate, polyethylene, polypropylene, polylactic acid, poly(D,L-lactide-co-glycolide), polycarbonate, cyclic olefin copolymers, silicon, glass etc.
Different manners of functionalizing these types of surfaces with the above mentioned functional groups are shown in the examples. In this sense, a thirteenth aspect of the invention refers to a process for functionalizing the sensing area of a microchip or device comprising a support or substrate, wherein said support or substrate comprises at least one channel in the substrate, the channel comprising an inlet, an outlet, and a flow-path connecting the inlet and outlet, wherein the inlet and outlet together define a midplane; and a portion of the flowpath travels transversely across the midplane, wherein the portion of the flowpath that travels transversely across the midplane includes a recognition site or sensing area for detecting a target analyte;
wherein if the support or substrate is made of:
Preferably, if the support of the microfluidic chip or device is made of a thermoplastic material the diazonium aryl compound is represented by formula I or II below:
wherein R is an alkyl group having from 1 to 15 carbon atoms or an ethylene; and
Z is a carboxylic group, an epoxy group, an amine group, a thiol group, an azide group, a halide, a maleinido functional group, a hydrazyde functional group, an aldehyde group or an alkyne group, preferably a carboxylic or epoxy group;
wherein R is an alkyl group having from 1 to 15 carbon atoms.
Preferably the diazonium component of formula I or II above is place or sited in the para or meta position with respect to the alkyl or ethylene component of any of these formulae. Examples of aryl amine compounds suitable for producing the diazonium aryl compound of any of formula I or II above are: 3-(4-Aminophenyl)propionic acid, 3-Aminophenylacetic acid, 4-Aminophenylacetic acid, 4-(4-Nitrophenyl)butyric acid or 4-(4-Aminophenyl)butyric acid.
In a further preferred embodiment of the thirteenth aspect of the invention, the surface of the microchip or device is further modified or functionalized with a chelating agent preferably selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt, nitrilotriacetic acid (NTA) metal (II) salt, iminodiacetic acid (IDA) metal (II) salt, Ethylenediaminetetraacetic acid (EDTA) metal (II) salt, diethylenetriaminepentaacetic acid (DTPA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+. This is accomplished by direct reaction of the chelating agent with any of the activated functional groups referred to above, wherein:
Preferably, the support is made of a thermoplastic material and the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC).
More preferably, the support is made of a thermoplastic material, the aryl amine compounds contain carboxylic groups activated via esterification with N-Hydroxysulfosuccinimide salt (Sulfo-NHS) catalyze by (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and the chelating agent is selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt or nitrilotriacetic acid (NTA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+, preferably Cu2+.
As illustrated in the examples, activation of the microchip's surface with a chelating agent such as ANTA metal (II) salt is especially advantageous to achieve an oriented configuration of the antibody resulting in an improved sensing platform.
Kit or Device Having a Sensing Area Functionalized for an Antibody Oriented Immobilization.
Lastly, it is noted that, as illustrated in the examples, by functionalizing the sensing area of any support, not necessarily the support of a microchip or device, such as glass, so that a capture biomolecule, such as an antibody, has an oriented configuration provides clear advantages for the detection of an analyte in a sensor system which uses the Heatsens technology.
Thus, a fourteenth aspect of the invention refers to a kit or device comprising a support or substrate, wherein said substrate or surface may be made from a variety of materials such as thermoplastic materials, silicon, metals, or carbon; wherein said support or substrate includes a recognition site or sensing area for detecting a target analyte; and wherein said recognition site or sensing area is functionalized with a chelating agent.
In a preferred embodiment of the fourteenth aspect of the invention, the chelating agent is preferably selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt, nitrilotriacetic acid (NTA) metal (II) salt, iminodiacetic acid (IDA) metal (II) salt, Ethylenediaminetetraacetic acid (EDTA) metal (II) salt, diethylenetriaminepentaacetic acid (DTPA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+. The chelating agent functionalizes the support by direct reaction of the chelating agent with an activated functional group, wherein:
It is noted that in the section entitled “PROCESSES FOR FUNCTIONALIZING THE SENSING AREA OF A MICROCHIP OR DEVICE SUITABLE FOR CARRYING OUT IMMUNOASSAYS BY DETECTING AN ANALYTE BY USING THE HEATSENS TECHNOLOGY”, we described how to functionalize different supports or surfaces with any of the organic functional groups mentioned through-out the present invention.
Preferably, the support is made of glass functionalized via self-assembly with organo-functional alkoxysilane molecules carrying one or more carboxylic groups or epoxy groups or amine groups or thiol groups or azide groups or halides or maleinido functional groups or hydrazyde functional groups or aldehydes or alkynes groups; wherein said functional groups have been optionally activated and directly reacted with a chelating agent, preferably with a chelating agent selected from the list consisting of: Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA) metal (II) salt or nitrilotriacetic acid (NTA) metal (II) salt, wherein metal (II) salt is understood as a salt of a divalent metal such as Cu2+, Ni2+ or Co2+, preferably Cu2+.
In a further preferred embodiment of the fourteenth aspect of the invention or of any of its preferred embodiments, the recognition site or sensing area may comprise:
Preferably, said recognition molecule can be selected from, but not limited to, the list consisting of: peptides, polysaccharides, toxins, protein receptors, lectins, enzymes, antibodies, antibody fragments, recombinant antibodies, antibody dendrimer complexes, nucleic acids, (DNA, RNAs), peptide nucleic acids (PNAs), molecular imprints. Preferably, said recognition molecule is an antibody, a fragment thereof or a recombinant antibody.
In a fifteenth aspect of the invention, the kit or device of the fourteenth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In a sixteenth aspect of the invention, the kit or device of the fourteenth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
In a seventeenth aspect of the invention, the kit or device of the sixth aspect of the invention or of any of its preferred embodiments, may further comprise at least one of the following elements:
Preferably the kit or a device of any of the fifteenth to seventeenth aspects of the invention, further comprises a device capable of detecting the heat generated by the metal nanoparticles when they are irradiated with the external light source selected from the list consisting of infrared cameras or thermopiles.
Lastly, an eighteenth aspect of the invention refers to the in vitro use of the kit or device of any of the fourteenth to seventeenth aspects of the invention for detecting an analyte as a result of the heat generated by metal nanoparticles when they are irradiated with an external light source.
The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the group.
Fabrication of the Microchip.
Sandwich immunoassays for the detection of pathogens such as Salmonella and Campylobacter, allergens such as the Ara h1, and other protein molecules such as albumin and collagen, have been implemented in microfluidic chips in the present invention. They were properly sketched and fabricated, as displayed in
The final chip design includes three dedicated inlets to allow a straight forward insertion of the reagents (see
The layout of the microfluidic chip, was originally conceptually designed for the specific detection of pathogens present in the poultry sector, even though the microchip referred to herein was also successfully applied for the detection of other biomolecules such as the Ara h1, collagen and albumin. In this sense, the present invention is not limited to the specific layout of the microfluidic chip described herein.
Reagents for the Different Functionalizations of the Microchip
4-(4-Aminophenyl)butyric acid (PhBut), sodium nitrite (NaNO2), hypophosphorous acid (H3PO2, 50 wt. % in H2O), (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS), Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (ANTA), copper (II) sulfate, 20 mM HEPES buffer pH 8.0, 2.5 N NaOH solution, 1 N HCl solution, absolute ethanol (EtOH), distilled Type-I water (>18.2 Mohm-1). Streptavidin Ref 7100-05 Lote A2805-PA05H. 2% solution of 3-(2,3-epoxypropoxy)propyltrimethoxysilane in dry toluene. 10 mM carbonate buffer pH 10.8. 10 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 5. Piranha solution (H2SO4:H2O2 3:1)
Reagents for the Detection of Salmonella thiphymorium
a. Capture antibody: Anti-Salmonella typhimurium 0-4 antibody [1E6] ab8274-Abcam 2 mg/mL.
b. 5 μg/mL disuelto en PBS 1×.
c. Blocking: TBS-T 0.1%+BSA 5%.
d. Antigen: BacTrace Salmonella typhimirium positive control Ref 50-74-01-KPL. Cell count: 3×109 CFU/mL.
e. Detection antibody: Anti-Salmonella antibody (Biotin) ab69255-Abcam 4 mg/mL
f. Dilution 1/5.000 dissolved in TBS-T 0.1%+BSA 5%.
Reagents for the Detection of Campylobacter jejuni
a. Capture antibody: Anti-Campylobacter jejuni antibody ab155855 Lote: GR146930-4 0.1 mg/mL.
b. Dilution 1/20=5.0 μg/mL PBS1×.
c. Antigen: BacTrace Campylobacter jejuni positive control Ref 50-92-93. Lot 140513-KPL Cell count: 4.64×108 CFU/mL.
d. Detection antibody: Anti-Campylobacter jejuni antibody-Biotin ab53909 Lot GR93260-3.
e. Dilution 1/1000 TBS-T 0.1%+BSA 5%.
Reagents for the Detection of Ara h1
a. Capture antibody: Monoclonal antibody 2C12 Mouse IgG1 Lot: 30083 2.7 mg/mL
b. Dilution 1/500=5.4 μg/mL PBS1×.
c. Allergen Standard nArah1 Ref EL-AH1-Standard. Lot 38018 20.000 ng/mL.
d. Detection antibody: Monoclonal Antibody 2F7 Mouse IgG-Biotinylated Lot 36069.
e. Dilution 1/1000 PBS-T 0.1%+BSA 5%.
f. Dilution 1/2000 PBS-T 0.1%+BSA 5%.
Reagents for the Detection of Albumin and Collagen
a. The OVA polyclonal antibody: Goat Anti-Rabbit IgG H&L (Biotin), Abcam ref: ab6720
b. Monoclonal Anti-chicken egg albumin (ovalbumin) antibody produced in mouse, Sigma ref: A6075
c. Mouse monoclonal to collagen I, GeneTex ref: GTX26308
d. Rabbit polyclonal to collagen I (Biotin), Genetex ref: GTX26577
Surface functionalization with carboxylic groups is obtained by covalent grafting of the aryl radical of diazotated PhBut (Scheme I) generated by both chemical reduction (H3PO2) and UV radiation that bonds to the chamber chip's surface (Scheme II).
1. Diazotation of PhBut
Diazotated PhBut is obtained in situ previous to its use in an ice bath by dissolving the amount of NaNO2 to reach a 0.3 M final concentration, in a 0.1 M PhBut solution prepared in 0.5 M HCl. This mixture is held at 4° C. for 10 min before use in surface modification.
2. Covalent Grafting of Diazotated PhBut.
Prior the surface modification, the chips are rinsed with ethanol and dried. Then, they are irradiated for 15 minutes with ultraviolet (UV in the range between 305 and 395 nm) light by exposing under UV-lamp (8 W) at wavelength of 365 nm. Diazotated PhBut solution just prepared as above described is mixed with H3PO2 acid solution to reach a 0.16 M final concentration and drop casted on the chip's chambers. They are again placed under UV-lamp and irradiated for 30 min at a wavelength of 365 nm. Finally, the modified chips are removed from the lamp and extensively rinsed with absolute EtOH.
NTA-Cu(II) surface modification is accomplished by activating carboxylic groups and direct reaction with primary amine (—NH2) of ANTA via EDC/SNHS-mediated amidation (Scheme III).
Activation of the carboxylate groups of the surface-modified chambers and subsequent amidation of the NHS-esters with the ANTA-Cu(II) complex is performed in several steps. Firstly, a 20 mM SNHS and 10 mM EDC solution is prepared by dissolving sulfo-NHS reagent in distilled Type-I water and transfer to EDC reagent. This solution containing the reagents is drop casted on the chip's chambers and allowed to react for 1 h at room temperature. Following, the chips are rinsed with distilled Type-I water and incubated in a solution of 25 mM ANTA in 10 mM sodium bicarbonate solution, pH 10 overnight to introduce the chelate. Finally, after removing the reagent excess by washing with water and dried, nitrile-tri-acetic-Cu(II) complex (ANTA-Cu2+) is formed on the surface by incubation of the chip's chambers in a 100 mM copper (II) sulfate aqueous solution for 3 hours. The chips are again wash and dried being ready for antibody immobilization.
The surface modification with others NTA-M2+ complexes can be also accomplished following the same procedure as for NTA-Cu(II) employing instead of CuSO4, the corresponding metal salt (CoCl2, NiSO4 or NiCl2) in similar concentrations as above described. The binding affinity of the NTA-chelated metal atom towards histidine-tagged proteins and antibodies follows the order Cu(II)>Ni(II)>Co(II).
Two types of bio-functionalization of glass surfaces have been performed, covalent non-oriented and oriented immobilization. To activate glass supports, surfaces are cleaned with piranha solution for 1 hour at room temperature in an orbital shaker. Subsequently, slides are rinsed with milli-Q water and dried. Then, 2% solution of 3-(2,3-epoxypropoxy)propyltrimethoxysilane in dry toluene is added overnight at room temperature in an orbital shaker onto the activated glass supports. Afterwards, slides are washed thoroughly with toluene and 10 mM carbonate buffer pH 10.8. After drying the slides, glass supports are incubated with 25 mM NTA for 3 hours at room temperature in an orbital shaker. Later, glass supports are washed extensively with 10 mM carbonate buffer at pH 10.8.
In order to have an oriented immobilization, NTA-surfaces are incubated overnight with 100 mM CuSO4 in aqueous solution at room temperature for complexation environment. Then, slides are washed with milli-Q water. For a non-oriented covalent immobilization, NTA-surfaces are loaded with 50 mM EDC and 75 mM SNHS in 10 mM MES pH 5 for 45 min at RT for further carboxyl group activation. Then, surfaces are washed with 10 mM MES pH 5.
1. Physical Absorption
Prior to the antibody surface modification, the chips are rinsed with EtOH and dried. Then 5 μl of 5 μg/ml of capture antibodies in PBS 1× are casted only onto the surface of the Microfluidic chamber (sensing area) inside the microfluidic channel and incubated at 37° C. for one hour.
The surface is rinsed with PBS 1× and incubated over night at 4° C. with blocking buffer (BSA 5% in PBS1×, 0.1% tween).
The surface is washed and the chip is assembled with the upper part (PMMA) and connected to the peristaltic pump.
2. Carboxylated-Functionalized Microfluidic Chamber Chip Surface Modification with Capture Antibodies: Covalent Antibody Immobilization
The activation of the carboxylate groups of the surface-modified chambers and subsequent amidation of the NHS-esters with the capture antibodies is performed in a two-step process as described as follows:
After the covalent immobilization of the capture antibodies and the blocking of surface with BSA 5% in PBS1×/0.1% Tween for 1 hour at 37° C., the chip is connected to the peristaltic pump and each channel is rinsed with washing buffer using a flow rate of 300 μl/min for 4 minutes.
3. Modification of Nitrilotriacetic-M(II) (Cu2+, Ni2+, Co2+) Complexes Functionalized Microfluidic Chamber Chip with Capture Antibodies: Oriented Antibody Immobilization
Modification with capture antibodies onto a NTA-M(II) (Cu2+, Ni2+, Co2+)-functionalized Microfluidic chamber chip is carried out in a single step, as described as follows: 5 μl of 5 μg/ml of capture antibodies are deposited only on the surface of the sensing area of the microfluidic channel and incubated for at 37° C. for one hour.
Then the surface is rinsed and incubated over night at 4° C. with blocking buffer (BSA5% in PBS 1×, 0.1% tween). Following, the surface is rinsed and the chip is assembled with the upper part (PMMA) and connected to the peristaltic pump.
In the following we illustrate different immunoassays implemented in the microchip referred to in the materials and methods for the detection of Salmonella. In addition, we have also compared the results obtained with these methods.
1. Direct Immunoassay for Salmonella Detection: Temperature Increment of First Test Using Chips Directly Functionalized with Two Different Dilutions of Salmonella
The unmodified fabricated microfluidic chip illustrated in the materials and method was used for testing the direct immobilization of two dilutions of salmonella.
10 μl of 60000 CFU/ml and 20000 CFU/ml (600 and 200 CFU in total on the surface, respectively) of Salmonella T. were adsorbed on the detection surface. After the direct immobilization of the pathogen, the surface was blocked with BSA and left to react with biotinylated detection antibodies. Finally, they were washed and further reacted with streptavidin-AuNanoprisms solution.
In order to test the specificity of the immunoassay, the following control experiments were performed: 1) NC1=absence of Salmonella, surface blocked with BSA 5%; 2) NC2=absence of biotinylated detection antibody; 3) NC3=absence of streptavidin-AuNPrisms.
In
In absence of biotinylated detection antibody (NC2) there is an insignificant increment in temperature as in absence of strepavidin@AuNPrism (NC3).
The increment of temperature is proportional to the amount of salmonella's CFUs. These results indicate the suitability of this material for the fabrication of the microfluidic chip and its application for HEATSENS. Moreover, the results envisage the possibility of immobilizing salmonella at different CFU dilutions directly onto a microfluidic chip and build a calibration curve.
2. Direct Immunoassay for Salmonella Detection: □Temperature Increment of First Test of Direct Immobilization of Salmonella and Detection of Two Different Dilutions of Salmonella on a Microfluidic Chip. Calibration Curve Test Construction.
10 μl of different concentrations (CFU/ml) of salmonella T, in a range between 0 and 240000 CFU/ml, were directly adsorbed onto the microfluidic chip and detected with biotinylated antibodies anti-salmonella to measure the increment of temperature due to the presence of different concentrations of salmonella. Then the strepavidine@AuNprism interacted with the antibodies and every single sensing area was irradiated with an IR laser. The temperature of each chamber was measured, and the increment of temperature calculated.
The increase of temperature measured was due to the increased amount of CFUs directly adsorbed onto the surface of microfluidic chip.
3. Sandwich Immunoassay for Salmonella Detection: Q Temperature Increment of First Test of Sandwich Immunoassay Detection of Two Different Dilutions of Salmonella on a Microfluidic Chip
Once shown that the microfluidic chip is suitable to be applied to the HEATSENS technology, we performed a sandwich immunoassay for the detection of the selected pathogen by using a microfluidic chip. For this purpose, each micro-chamber of the microchip was functionalized with capture antibodies anti-salmonella by direct adsorption of (5 μL) 5 μg/ml of capture antibodies anti-salmonella onto the surface. Then, the salmonella's capture event was carried out in fluidic mode, as well as the detection and the interaction with the streptavidin-AuNprism, injecting 1 ml of sample, in each channel.
The assay was carried out with 2 different concentrations of salmonella's CFU/ml, 200000 CFU/ml and 240000 CFU/ml diluted in buffer phosphate, respectively.
The trend of the calibration curve is not linear, indicating a saturation of the signal due to the presence of high amount of nanoprisms interacting with the analyte. The detection of the two unknown concentrations of salmonella was calculated from the exponential equation, where the values concur with the curve with an adj. R-Square equal to 0.98843.
Once shown the effectiveness of an immunoassay in a sandwich format, we tried to improve the limit of detection of salmonella t., by decreasing the concentration of the pathogen in doped buffer.
1500 CFU/ml of salmonella T. in PBS 1× was the first lower concentration detected in the first trial.
1 ml of sample was injected in the channel with a flow of 200 μl/ml. After injecting the sample, the channel was washed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/min for 4 min. Then the detection antibodies were left to interact with its antigen using a 200 μl/ml for 2 minutes. The channel was rinsed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/min for 4 min. The streptavidin@AuNPr were injected into the channel. The flow was 200 μl/ml for 2 minutes. The channel was rinsed with washing buffer (BSA 0.5% in PBS1×, 0.1% tween), using a flow of 300 μl/mim for 4 min and dried.
The temperature increment due to the presence of salmonella was higher than all negative controls, even though different from the expected value: the positive values of increment of temperature of the negative controls indicated non-specific interactions between the reagents within the immunoassay. The non-specific interactions can be associated to an uncompleted functionalization and blocking of the surface or to an inappropriate flow rate during the immunoassay. In this way, by keeping constant the surface antibody functionalization and modifying the flow rate during the immunoassay, it was possible to improve the limit of detection of salmonella and the signal due to the background, as shown in
The same experiment was carried out using a real food sample, 25 μg of chicken meat in 225 ml of peptone pre-enrichment culture media, doped with salmonella at different CFUs. The capture antibodies were adsorbed onto the microfluidic chip, and the surface blocked with 5% BSA in PBS1×-01% Tween, using a flow rate of 150 μl/min.
Then, the washing was carried out using a flow rate of 250 μl/min, by using a washing buffer.
The capture of salmonella in 1 ml of real sample, as well as the detection with biotinylated detection antibodies, and the interaction with streptavidin@nanoprisms was performed by using a flowing at a flow rate of 15 μl/min.
The results of the immune analysis carried out in the microfluidic chip are shown in
After building the calibration curve, measuring the increment of temperature due to the known different concentrations of salmonella, the unknown concentration of pathogen in the real sample was determined from the calibration curve (
The higher increment of temperature of the samples doped with salmonella, clearly indicates that HEATSENS is suitable for the ultrasensitive detection of few CFUs of bacteria in complex matrices such as the 25 g of chicken meat in 225 ml of peptone.
The increment of temperature due to the presence of salmonella in a real sample is slightly different from the one in buffer phosphate, because of presence of high amount of meat proteins which affect the specific interaction of the bacteria with the antibodies.
4. Sandwich Immunoassay for Salmonella Detection: Effect of Covalent Immobilization of the Capture Ab on Microfluidic Chip
The modification of a microfluidic chip surface with carboxylic end group can be used to immobilize covalently capture antibodies by formation of stable amide bonds with their primary amines via EDC/sulfo-NHS reaction.
In this sense, the surface of each micro-chamber, previously activated with 10 mM EDC and 20 mM sulfo-NHS, was functionalized with 20 μl of 5 μg/ml of capture antibodies. After the covalent immobilization of the capture antibodies and the blocking of surface with BSA 5% in PBS1×/0.1% Tween for 1 hour at 37° C., the chip was connected to the peristaltic pump and washed with washing buffer using a flow rate of 300 μl/min for 4 minutes. 1 ml of 30 CFU/ml of Salmonella T, were allow to flow inside the microfluidic channel for 1 minute at a flow rate of 150 μl/min, then the channel was washed with a buffer solution using a flow rate of 300 μl/min for 4 minutes. 400 μl of biotinylated detection antibodies were then flowed inside the channel.
The results depicted in
5. Sandwich Immunoassay for Salmonella Detection: Oriented Immobilization of Capture Antibodies Through Metal-Chelation on Microfluidic Chip.
Oriented immobilization of antibodies through metal-chelation constitutes an optimal and versatile method as shown herein. Immobilization is accomplished through the metal-chelation to histidine-rich metal binding site in the heavy chain (Fc) of the antibody or to poly-His-tag sequence fused in proteins. Since the metal binding site is either in the C- or N-terminus, antibodies and His-tagged proteins bound in this fashion to the surface are oriented with the combining site directed away from the surface thus allowing maximal antigen binding or a favourable protein orientation. Furthermore, oriented immobilization through metal-chelation also results in a stable antibody immobilization since binding constants for metal-chelation immobilization are very high due to the combination of the chelate effect of histidine binding, and target binding of multiple metal-chelate groups. Dissociation constants are estimated to be between 10−7 to 10−13 M−1. For many applications, this provides binding strengths comparable to antigen-antibody interaction. On the other side, experimental conditions of antibody attachment for oriented immobilization of antibodies through metal-chelation are milder than those employed for covalent oriented immobilization procedure. As an advantage, the antibody binding to the chelate could be also modulated as convenience to be reversible or irreversible. In addition, it is also more versatile since it can be also employed for immobilization of his-tagged recombinant proteins.
In order to achieved an oriented immobilization of the capture antibodies, the microfluidic chamber chips were functionalized with metal-chelate complexes in a stepwise modification of their surface. Firstly, the surfaces were functionalized with aryl amine compounds containing carboxylic groups such as for example 3-(4-Aminophenyl)propionic acid, 3-Aminophenylacetic acid, 4-Aminophenylacetic acid or 4-(4-Nitrophenyl)butyric acid. For this specific example we used PhBut, even though for the immobilization of different biomolecules, it would be more appropriate the use of aryl amine compounds carrying different lengths of n-alkyl carboxylic acids in a range between 2 and 16 carbons.
Carboxylic groups introduced by covalent grafting of the aryl radical of diazotated PhBut (Scheme II) were activated by esterification with SNHS catalyzed by EDC to facilitate the covalent linkage of the ANTA-M(II) (Cu2+, Ni2+, Co2+) complex (Scheme III) through the free amino groups. Then, they were incubated with 20 μl of 5 μg/ml of capture antibodies. The resulting NTA-M(II) complex termination contains two free coordination sites occupied by water molecules to be replaced by histidine residues of capture antibodies giving rise to their oriented immobilization. Later, the chip was connected to the peristaltic pump and washed with washing buffer using a flow rate of 300 μl/min for 4 minutes. 1 ml of 30 CFU/ml of salmonella T., was allow to flow inside the microfluidic channel for 1 minute at a flow rate of 150 μl/min, then the channel was washed with buffer using a flow rate of 300 μl/min for 4 minutes. 400 μl of biotinylated detection antibodies was then flowed inside the channel.
Interestingly, the temperature increment due to the presence of Salmonella for this type of immobilization was higher than those obtained for the respective controls and even higher than those obtained in previous results for direct adsorption and covalent immobilization. In this sense, a comparative study between the different immobilization methods was carried out. The comparison of the different strategies of antibody surface functionalization is displayed in the
The advantageous antibody oriented immobilization shown in example 6, was tested for the detection of salmonella in a real sample. The result is reported in the
The temperature increment, due to the presence of salmonella in the real sample on an oriented antibody immobilized microfluidic chip surface, was also higher than those obtained for the respective controls.
After building the calibration curve, the measurement of the increment of temperature due to the known different concentrations of salmonella and to the unknown concentration of pathogen in the real sample was determined, as reported in the
The increment of temperature due to the presence of the theoretical number of CFU/ml used to dope the real sample, agrees with the number of CFUs of the calibration curve.
The established protocol for the capture antibody oriented functionalization of microfluidic chamber, together with a sandwich immunoassay, was used for the detection of a pathogen different from Salmonella such as Campylobacter jejuni in order to demonstrate the universality of this technology.
Campylobacter jejuni is one of the four bacterial pathogens, together with Salmonella spp., Listeria monocytogenes (L. monocytogenes), and Escherichia coli (E. coli) O157:H7, estimated to account for approximately 67% of food-related deaths (Mead et al., 1999). Screening for Campylobacter is routinely carried out globally with different quantification methods which are available for the detection of this pathogen in food products, such as culturing, microscopy, enumeration methods and bio-chemical test PCR, immunoassays (Yang et al., 2013). Some of the aforesaid methods are sensitive and rapid but suffer from setbacks such are the fact that they are expensive, require extensive sample preparation, have poor selectivity and are time-consuming.
Indeed, as for salmonella, since most poultry-based products are consumed within days from the production date, this presents a challenge for available methods as while the method is being performed the population is exposed to Campylobacter leading to out breaks of food borne illness (Che et al., 2001).
The immunodetection of C. jejuni using HEATSENS in a microfluidic chip provides a cost-effective, rapid, easy, sensitive and reliable diagnostic approach.
C. jejuni was purchased heat-killed and lyophilized. They were re-suspended in PBS at different dilutions, and used to generate the calibration curve for further detection of an unknown sample (
The combination of HEATSENS technology and the antibody oriented functionalization of the microfluidic chamber surface, allows achieving low LOD (Limit of detection) of campylobacter in Bolton culture media.
Compared with the sensing of Campylobacter J reported by Masdor et Al. (Masdor et Al. Biosensors and bioelectronics 78, 2016, 328-336), which describes the development of a sensitive QCM sandwich immunoassay with a detection of 150 CFU/ml of Campylobacter, HEATSENS allows a detection of this specific bacteria pathogen lower than 100 CFU/ml.
Furthermore, this limit of detection is reached immobilizing 210 fold less capture antibody on the surface, decreasing the background and lowering the cost of production of the chip.
To further illustrate the universality of the present methodology we performed the present example with a still further analyte.
Peanuts (Arachis hypogaea) are one of the allergens most frequently associated with severe allergic reactions, including life-threatening food-induced anaphylaxis. According to the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA 2004, Public Law 108-282, Title II) in the United States, and the Directive 2000/13/EC, as amended by Directives 2003/89/EC and 2007/68/EC, in the European Union, the presence of peanut in a food product has to be declared on its label.
The current reference method for detecting food allergens is the ELISA, even if there are also other analytical methods such as HPLC, capillary electrophoresis (CE), methods with laser-induced fluorescence (LIF) detection, enzyme linked immune affinity chromatography (ELIAC), size exclusion chromatography, and SPR. The determined LOD of ELISA is showed in the
The combination of HEATSENS technology and the antibody oriented functionalization of the microfluidic chamber surface, allows to achieve lower LOD of Ara h1 in PBS using the same pair of capture and detection antibody (
HEATSENS was thus successfully employed, in combination with oriented functionalized microfluidic surface in a bioassay to detect Ara h1.
The biosensor detection limit for Ara h1 was improved by one order of magnitude (LOD<0.4 ng/ml) compared with commercial ELISA kits (LOD Z10 ng/ml), and several orders of magnitude compared with other detection methods such as the SPR (J. Pollet et al./Talanta 83 (2011) 1436-1441).
The characterization of historic paints' binders still relies on conventional molecular biology methodologies that were developed decades ago and which have been substituted by more sensitive, specific, inexpensive and faster methodologies, taking advantage of the benefits of the emerging nanotechnology world.
HEATSENS was applied to the detection of collagen and albumin, two of the most used binders in pre-Renaissance paintings, illuminated manuscripts and sculptures in a microfluidic chip. This example again further illustrates the universality of the present methodology
1. Direct Immunoassay for the Detection of Albumin Absorbed onto a Microfluidic Chip Chamber Surface.
We implemented a direct immunoassay for the detection of Albumin. For this purpose, albumin as positive control (PC1), two micro-samples: one of albumin in powder from Zacchi® (sample 4) and another from glair painted on a glass surface exposed to the air for 1 year and a half (sample 5), samples were directly immobilized onto the microfluidic chamber surface. After the immobilization, the surface of the chip was blocked with milk in PBS 3 mg/mL, covering the chip surface, for 1 hour (at least) at 37° C. and shaking.
In order to test the specificity of the immunoassay, the following control experiments were performed: 1) NC1=absence of albumin and 2) NC2=absence on detection antibodies.
The results of the direct immunoassay, carried out in the microfluidic chip following the settle protocol, is depicted in
The result demonstrates that HEATSENS, employed in combination with functionalized microfluidic surface in a bioassay, is able to detect Albumin in pigments. The present sensing methodology offers also the possibility of albumin quantification in complex matrices as the pigments are.
2. Sandwich Immunoassay for Detection of Collagen Using Capture Antibody Covalently Immobilized on Amicrofluidic Chip Surface
The detection of collagen, was also implemented by using an immunoassay in a sandwich format.
The capture antibodies were immobilized onto the microfluidic chip surface using the already described immobilization protocol, and two micro-samples: one from rabbit skin glue in water (10% w/w) (sample 4) and another micro-sample from a paint made by a mixture of glue+CaCO3 painted over 40 years ago (real sample) that was recognized by the detection antibodies, were allow to flow inside the microfluidic chip.
In order to test the specificity of the immunoassay, the following control experiments were performed: 1) NC1=absence of collagen and 2) NC2=absence on detection antibodies.
The combination of HEATSENS technology and the antibody functionalization of the microfluidic chamber surface, allows to identify collagen in real samples.
The result of the collagen detection, using HEATSENS technology in a microfluidic chip is showed in the
The result demonstrates that HEATSENS, employed in combination with functionalized microfluidic surface in a sandwich immunoassay, is able to detect collagen in pigments. The present sensing methodology offers also the possibility of collagen quantification in complex matrices as the pigments are.
The following protocol was found to be especially suitable for sandwich immunoassays using a microfluidic device and the Heatsens technology:
Oriented immobilization methodology through functionalization with metal-chelate on microfluidic chip can be extended to other types of surfaces such as metal (iron, cobalt, nickel, platinum, palladium, zinc, copper and gold), carbon (graphene, diamond, nanotubes, nanodots) and silicon surfaces. Grafting of diazonium aryl derivatives containing carboxylic groups can also be accomplished on these surfaces being a platform for a further stepwise functionalization with the metal-chelate layer.
It is also possible to functionalize other surfaces such as Polydimethylsiloxane (PDMS) and glass by covering the surface through self-assembly with organo-functional alkoxysilane molecules carrying a carboxylic acid function or epoxy groups. In this way, a study was carried out in a glass surface functionalized with epoxy groups by silanization and further introduction of a metal-chelate layer (NTA-Cu2+). The metal-chelate functionalized glass surfaces were assayed for both oriented and non-oriented covalent immobilization of biomolecule and employed the sandwich immunoassays to detect analytes in a sensitive way.
Simple glass surface modification was carried out in four steps. For the first step, the activation of glass supports was performed to remove all the organic residues in order to graft the epoxysilane on the surface. In the second step, the functionalization with epoxysilane was done with dry toluene to avoid gel formation of the silanes. The epoxy groups on the surface guarantee an efficient reaction with the amine group of the NTA at pH 10.8, where the amine of the NTA opens the epoxy group in a high molar ratio. And finally, in the last step, supports were incubated with 100 mM of CuSO4 in order to chelate the metal ion onto NTA moiety to orient the analyte.
Once glass slides were functionalized with NTA-Cu2+, an immunoassay for the detection of salmonella using HEATSENS technology was carried out (by using an oriented immobilization). For comparison, other methods of immobilization such as direct adsorption and covalent flat-on antibody immobilization were also assayed. The results of the different strategies of antibody surface functionalization are displayed in
This figure again demonstrates that the oriented immobilization of capture antibodies through metal-chelation provides the best results not only in terms of a high increment of temperature due to the presence of salmonella but also by providing a null signal due to nonspecific interactions (background). Thereby indicating the correct functionalization strategy as a crucial step to obtain an optimal antibody attachment in a favorable orientation, while avoiding nonspecific adsorption of HEATSENS labels (gold nanoprisms).
Glass surface functionalization is fast, easy, simple and inexpensive and can be used for different types of biomolecules.
The important advantage of the sensing setup of HEATSENS using microfluidic chips for analyte capture is that all components are suitable for being assembled and miniaturized in a number of different ways, one of these being the one shown in
Two possible configurations of the sensor system are mentioned below:
1. Thermal sensor behind sample; and
2. Thermal sensor in front of sample
In this sense, we can place the laser and thermopile (thermal sensor) in the same plane, with the thermopile pointing at the sample, tilted lightly upwards, or in different planes. The inclination is due to the saturation of the thermopile. When the laser beam irradiates directly to the thermopile, every temperature value reaches the maximum and the measurement is not valid. The thermopile has a FOV of 100×400 and the cameras, where the reaction takes place, are 3 mm high, 5 mm width. This results in an optimal distance between sample and thermopile of 17 mm to cover the camera; to ensure the measurement it is set at 20 mm.
If we place the laser and the thermopile in the same plane and we measure from behind, the sample is located with the thinner width next to the thermopile, so that the heat detected would not spread out and we can get the total information.
The results registered by using the configuration shown in
However, the laser and thermopile can also be place in different planes. Once again the thermopile will be pointing to the sample, vertically tilted (≈40°) to avoid the laser irradiation (
Measuring in front of the sample requires that the thinner width is on the thermopile and laser side. Here the laser irradiates in a focused manner, the light goes through a thinner part of the μfluidic chip and irradiates the sample.
In
The presented results were achieved using the Ventus laser system but considering the characteristics of the HEATSENS technology, also other NIR light source could be used such as the laser diode and LED.
For sensing applications, the immobilization of the recognition biomolecule on the support where it occurs the sensing, must be as stable as possible, oriented and with a high-yield, to provide a high sensitivity to the sensing platform.
For HEATSENS sensing platform, as reported in the present specification, the chemistry has been modified for the specific oriented immobilization of capture antibodies used for the implementation of the sensing platform. There are two key factors for the oriented immobilization of antibodies on surfaces through the metal-chelation of NTA-M2+ to the histidine-rich metal binding site present in the antibody heavy chain (Fc):
The first key factor of the HEATSENS sensing platform is the NTA-metal chelates employed. In this sense, we have assayed the immobilization of antibodies on gold nanoparticles functionalized with NTA-metal chelates: NTA-Cu2+ and NTA-Co2+ employing anti-HRP and anti-CD3, respectively, to demonstrate the unique methodology to be used to reach high sensitivity of the HEATSENS sensing platform.
The amount of immobilized antibody was calculated by measuring the protein remaining in the supernatant before and after every step in the immobilization process. Samples were withdrawn and analyzed by SDS-PAGE. Gels (12%) were used and stained with silver.
As it can be seen in the SDS-PAGE gels (
The antibody immobilization on gold nanoparticles functionalized with NTA-Co2+ and NTA-Cu2+, was also evaluated by incubation with HRP and following measurement of its enzymatic activity. As it can be seen in
The high affinity of antibodies for copper in comparison to other bivalent metals is also demonstrated using commercial strips of flat surface functionalized with copper ions and nickel ions (2D system). In this sense, antibody molecules against HRP were immobilized on these functionalized metal-chelate surfaces and the presence of captured HRP was quantified by a colorimetric immunoassay. As it is shown in
These results make evident the higher binding capacity of the antibodies oriented immobilized onto surface activated with copper chelate when compared to Ni. The same experiment has been carried out using asymmetric gold nanoparticles as label, for HEATSENS sensing detection (please refer to
The higher antibody capture efficiency of the copper chelated surface is also established by using the HEATSENS detection methodology. In this sense, the increment of temperature nearly duplicated when 10 μg/mL of anti-HRP were immobilized on Cu ions in comparison to Ni ions.
All these results demonstrate the importance of the metal employed for the oriented immobilization of antibodies on surfaces through the metal-chelation of NTA-M2+.
Another key factor, that has a strong influence for the oriented immobilization of antibodies on surfaces through the metal-chelation of NTA-M2+ to the histidine-rich metal binding site present in the antibody heavy chain (Fc), is the metal-chelate surface density, which affects the yield of antibody immobilization. In order to demonstrate this fact, we performed an antibody-HRP immobilization study employing different coverages of NTA-Cu2+ using gold surface modified nanoparticles as a 3D system. These were obtained by varying the concentrations of EDC/sulfo-NHS as catalyzers for its incorporation.
Table 1 shows the enzymatic HRP activities of anti-HRP immobilized on gold nanoparticles functionalized with low and high surface coverages of NTA-Cu2+ and NTA-Co2+, respectively.
It is observed that only gold nanoparticles functionalized with a high surface coverage of NTA-Cu2+ after incubation with antibody-HRP gives practically full enzymatic activity associated to anti-HRP immobilization. On the contrary, no activity was observed for gold nanoparticles modified with a low concentration of NTA-Cu2+ or NTA-Co2+.
This demonstrates that antibody immobilization is significantly affected by the density of the metal-chelate on the surface, this being a crucial factor to be controlled.
The effect of the density of the active groups and of the metal on the immobilization of the capture antibodies onto the surface where the detection occurs, has also been evaluated on 2D surfaces. In this sense, we compared two different protocols. In particular, we used the protocol described in Chiu Wai Kwok et al, “In vitro cell culture systems for the investigation of the morphogen Sonic hedgehog (Shh), Dissertation, 16 Nov. 2011, wherein microchip surfaces were functionalized by UV irradiation (185 nm), leading to the generation of carboxylic groups.
We then formed the chelates by coordination of bivalent metals, such as Ni2+, with the carboxylic groups formed. The chelation was carried out using 40 mM NiSO2, which reacted with N2-N2-bis-(carboxymethyl)-L-lysine previously introduced via the amino terminal group on the COOH polymer surface. The metal modified surface was then used to immobilize a poly (6) hystidine tagged protein, in this specific case the ShhN protein.
Such protocol was compared to the HEATSENS surface functionalization, wherein in contrast to the above, the introduction of the carboxylic groups was accomplished by grafting of an organic layer using aryl diazonium salt chemistry and UV light (365 nm, 8 W); the carboxylic surface was then functionalized with 20 mM N2-N2-bis-(carboxymethyl)-L-lysine-25 mM CuSO4 via amidation catalyzed by 10 and 20 mM EDC sulpho-NHS, respectively. The two steps of chemical modification guarantee the creation of a homogeneous layer with a high density active groups.
Evaluation of the interfacial surfaces changes for both surfaces were characterized by Fourier Transform Infra-Red (FTIR) measurements. These were performed on a Spectrum One FT-IR Spectrometer equipped with the Universal ATR Sampling Accessory (Perkin Elmer).
The FTIR study of modified samples with NTA-Cu2+ functionalized by NIT procedure revealed the appearance of characteristic bands associated with the vibrational modes of amides (
CuSO4 forms a chelate with NTA in order to orient the antibody, where the ratio COOH:Cu2+ is 3:1, so 1 mol of Cu2+ corresponds to 3 moles of COOH of NTA. UV-Vis spectra of five points of calibration curve are shown in
Similar experiments were carried-out with surfaces modified with NTA-Ni2+ by using the methodology reported in Chiu Wai Kwok et al giving negligible absorbance values and therefore undetectable Ni2+ amount. These results further confirm the lower yield of the chelate functionalization employing Chiu Wai Kwok et al technology rather than NIT technology.
Differences in the antibody immobilization and its binding capacity on modified surfaces depending on the protocols of surface functionalization (NIT and Chiu Wai Kwok et al) were also analyzed. Antibody against HRP was immobilized on NTA-Cu2+ and NTA-Ni2+ chelate functionalized surfaces. Their binding capacity of immobilized antibody to capture the HRP is demonstrated by measuring the activity of the HRP on the surface, determined by a colorimetric method as well as by Heatsens detection.
This result is further confirmed by carrying-out a HEATSENS assay. In this sense,
Other approach to demonstrate the differences of the protocols and strength of NIT methodology in comparison to Chiu Wai Kwok et al have been the detection of the pathogen Salmonella employing the high sensitivity of HEATSENS sensing platform. Immobilization of antibody was assayed on both surfaces functionalized with NTA-metal chelates: NTA-Cu2+(following protocol developed in NIT) and NTA-Ni2+(following protocol reported in document Chiu Wai Kwok et al); a total amount of 1000 CFU of salmonella interacted with the oriented immobilized capture antibodies. Once onto surface, the biotinylated-detection antibodies detected the pathogen enabling the interaction with streptavidin-HRP (for the colorimetric assay) or streptavidin-nanoprisms (for the HEATSENS assay).
The results of HEATSENS assay relative to the detection of Salmonella on differently activated surfaces, are displayed in
| Number | Date | Country | Kind |
|---|---|---|---|
| 16382137.4 | Mar 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/057346 | 3/28/2017 | WO | 00 |
