DESC:FIELD OF THE INVENTION:
[0001] The present invention relates to the technical field of detection kits. More particularly, it relates to a multiplexed microfluidic assay device, for multi-sample detection. It also relates to a method of preparation of the assay device.
BACKGROUND OF THE INVENTION:
[0002] Pancreatic juice from the pancreas produces several digestive enzymes like pancreatic lipase, amylase, and protease which are essential for digesting sugars fats, and proteins. Serum amylase and lipase are critical biomarkers in diagnosing acute and chronic pancreatitis and other pancreatic disorders. Urinary trypsinogen-2 is another biomarker which enables detection of acute pancreatitis.
[0003] Traditionally, testing for these enzymes requires serum or plasma or urine collection followed by laboratory-based biochemical assays. These tests are expensive, required trained professionals and time consuming, wherein the exact period during which the peak of such biomarkers may be observed are likely to be missed due to various factors.
[0004] Lateral flow devices are known for detecting trypsinogen, but there remains a need for improved devices specifically tailored for detecting trypsinogen in urine with enhanced specificity, stability, and ease of use. Lateral flow test strips and similar formats have revolutionized rapid diagnostics in other domains (e.g., pregnancy testing, glucose monitoring), but there remains a gap in enzyme detection. The concentration of both serum amylase and lipase rises 3 times the normal value and level of urinary trypsinogen-2, beyond 50 ng/mL in the case of progression of acute pancreatitis together with intense abdominal pain radiating backward. Resultantly, any person with a sudden onset of abdominal pain, may be required with multiple tests to assess acute pancreatitis. It assumes significance as there are instances of misjudgement of gastritis pain as pancreatitis. In view thereof, there is required a test protocol, which combines the advantages of the two existing testing protocols, while also balancing the need for time and economic value.
[0005] Further, there is a growing need for a lateral flow test which combines the advantages of serum detection and trypsinogen detection in a single platform. There is a definite gap in the existing technology regarding the availability of a simple, rapid, low-cost, and reliable device that can detect the presence and/or levels of amylase and lipase and trypsinogen from body fluids such as whole blood, saliva, or urine.
[0006] The present invention seeks to overcome the drawbacks existing in the present technology by providing for a multiplexed microfluidic assay device.
OBJECTS OF THE INVENTION:
[0007] It is an object of the present invention to provide a multiplexed microfluidic test device.
[0008] It is another object of the present invention to provide for a test device with two separate zones, i.e., Zone 1(Enzymatic) and Zone 2 (Immunoassay).
[0009] It is yet another object of the present invention to provide for a test device wherein the Zone 1 comprises of two sub-test zones, i.e. A and L.
[0010] It is yet another object of the present invention to provide for a cost-effective test device to act as a point-of-care device.
SUMMARY OF THE INVENTION:
[0011] In an embodiment, there is disclosed a multiplexed microfluidic assay device, comprising a first zone device configured for colorimetric enzymatic assay to detect serum lipase and serum amylase; and a second zone device configured for colorimetric immunoassay to detect trypsinogen-2 biomarker using a lateral flow immunochromatography assay.
[0012] In this embodiment, the device [300] is separable into two zone devices, i.e., Zone 1 [100] and Zone 2 [200] at the marker [301].
[0013] In this embodiment, the zone devices [100, 200] are configured to receive a sample in the nature of bodily fluid selected from saliva, blood, serum, and urine.
[0014] In this embodiment, the zone devices [100, 200] provide visual indicators in the nature of colour gradation, as an indicator of presence of lipase, amylase, and trypsinogen-2.
[0015] In another embodiment, there is disclosed a method for preparing a multiplexed microfluidic assay device [300], the said method comprising fabricating detection zones [100, 200] on a qualitative filter paper using hydrophobic toner material. In this embodiment, fabrication involves printing the detection zones [100, 200] on the filter paper. Thereafter, the fabricated paper to heat treatment to induce hydrophobicity, resulting the assay device [300]. The heat-treated paper/assay device [300] is stored in a sterile environment further use.
[0016] In this embodiment, the detection zones [100, 200] are separated by a marking line [301] making it separable at the marking line [301].
[0017] In yet another embodiment, there is disclosed a method for preparing Zone 1 device [100] of a multiplexed microfluidic assay device [300], the said method comprising surface functionalization of sub-detection zones [102, 103] with biofunctionalized materials selected from Chitosan LMW, Polyethylene Glycol, PEG-Chitosan conjugate, gold nanoparticles (AuNP) and Chitosan nanopowder (CNP).
[0018] In yet another embodiment, there is disclosed a method for preparing Zone 2 device [200] of a multiplexed microfluidic assay device [300], the said method comprising assembling a lateral flow immunochromatography assay (LFIA) strip [201] by consecutively connecting and overlapping four components such as a sample pad [206], conjugate pad [205], test pad [207], and absorbent pad [203].
[0019] In this embodiment, the test pad [207] forms the base of the strip [201] The sample pad [206], the conjugate pad [205] and the absorbent pad [203] are assembled on the test pad [207].
BRIEF DESCRIPTION OF THE DRAWINGS:
[0020] The present invention will be described in more detail hereinafter with the aid of the accompanying drawings. The drawings are illustrative of one or more embodiments of the invention and do not in any manner limit the scope.
[0021] Figure 1 identifies a microfluidic test device, according to the present invention.
[0022] Figure 2 identifies a schematic representation of the test device, according to a preferred embodiment of the present invention.
[0023] Figure 3 identifies the activity of the test device zones corresponding to different lipase concentrations (69, 34.5, 17.25, 8.6, and 4.3) U/L onto different biofunctionalized paper devices, NC: non-coated, CNP: chitosan nanopowder, C: chitosan, PEG: polyethylene glycol, and PC: PEG-chitosan, (A) just after and (B) 5 minutes of adding lipase.
[0024] Figure 4 identifies standardization of different concentrations of p-NPB using patients and normal samples on the paper device. 4A is an Image and 4B is a graph showing quantification of the image using yellow colour intensity versus different pNPB concentrations on CNP-coated paper devices.
[0025] Figure 5 identifies the activity of the test device zones corresponding to different Amylase Concentrations (290, 145, 36.25, 2.26, and 0.56) U/L analysis in the (A) SI-BCG, and (B) SI-MR coated wells.
[0026] Figure 6 identifies amylase concentration analysis in the acute pancreatic serum sample (a) before, (b) immediate, (c) 5 min, and (d) 10 minutes of adding serum sample.
[0027] Figure 7 identifies dual detection zones in the device showing colour for decreasing concentration of Amylase/Lipase. (A) is the Front side and (B) is the Back side of the device.
[0028] Figure 8 identifies the components of the Immunoassay Strip for trypsinogen-2 detection
[0029] Figure 9 identifies optimization of capture antibody concentration (a) 2 min, and (b) 10 min of adding buffer solution.
[0030] Figure 10 identifies the Immunoassay test device showing colour intensity at the control line after the addition of buffer.
[0031] Figure 11 identifies Test devices1 & 2 showing test line for AP biomarker Human trypsinogen-2 in different concentrations (T1: 1 µg/ml, and T2: 0.5 µg/ml) before, 2 min and 5 min of adding of sample solution using (a) polyclonal Ab, and (b) monoclonal Ab as a capture reagent.
[0032] Figure 12 identifies optimization of the volume of the conjugate solution (test and control conjugate solution).
[0033] Figure 13 identifies the test devices showing test and control lines for AP biomarker Human trypsinogen-2 in different concentrations (0, 31.25, 62.5, 125, 250, 500, and 1000) ng/mL, 10 min of adding of sample solution.
[0034] Figure 14 identifies the test device showing test and control lines of positive control (1:1000 ng/mL), negative control (2: 0 ng/mL) and normal samples (S1: 20, S2: 22, S3: 39.7, S4: 31.3, and S5: 28.3) ng/mL, 10 minutes after adding of sample solution.
[0035] Figure 15 identifies the test device showing test and control lines of negative control (1: 0 ng/mL), (2: 500 ng/mL), positive control (1:1000 ng/mL), and patients’ samples (S1: 14, S2: 23, S3: 125, S4: 33 and S5: 74) ng/mL, 10 min after adding of sample solution.
[0036] Figure 16 identifies the test device showing test and control lines in a sample with trypsinogen-2 concentrations of 125 ng/mL performed at 3 independent days, 10 min after adding of sample solution.
DETAILED DESCRIPTION:
[0037] The following description illustrates various embodiments of the present invention and ways of implementation. The embodiments described herein are not intended to be limited to the disclosure and that the same is in no way a limitation. The invention may be embodied in different forms without departing from the scope and spirit of the disclosure.
[0038] Many other embodiments may be apparent to those of skill in the art upon reviewing the disclosure. Other embodiments may be utilized and derived from the disclosure, such that structural and logical substitutions and changes may be made without departing from the scope of the disclosure.
[0039] Specific dimensions and/or other physical characteristics relating to the embodiments disclosed herein are therefore not to be considered as limiting unless the claims expressly state otherwise.
[0040] Further, reference numerals are used only as an aid to explain the invention and they do not in any matter restrict the scope of the invention.
[0041] The present invention discloses a multiplexed microfluidic assay device. Further disclosed is a method of preparation of the assay device.
[0042] The assay device is configured to detect enzymes or biomarkers as per requirement. Throughout this specification, the invention may be referred to as the device, test strip, LFIA strip, strip, zones, zone devices etc. While the nomenclatures may be used interchangeably, the intent and object remain within the scope of the disclosure mentioned herein.
[0043] The device is configured to provide a multiplexed single platform for identifying biomarkers such as serum biomarkers (lipase and amylase) and trypsinogen-2.
[0044] The device has two distinct zones, i.e., Zone 1 (Colorimetric Enzymatic Assay Zone) and Zone 2 (Colorimetric Immunoassay Zone). In the Zone 1, the device detects two enzymes, i.e., serum lipase and serum amylase. In the Zone 2, the device detects trypsinogen-2 biomarker using a lateral flow immunochromatography assay.
[0045] Referring to Figure 1, the device [300] of the present invention is disclosed herein. As visualised in Figure 2, the present invention applies to both enzymatic and immunoassay detection in a single platform.
[0046] The device [300] architecture will be described hereinbelow, with reference to figures 1 and 2. The device [300] comprises of two zones, i.e., Zone 1 [100] and Zone 2 [200], separated by the marker or a marking line [301]. The marker [301] enables easy separation of the two zones [100, 200], resulting into two separate zone devices [100, 200], which are then capable of being used individually, as per requirement.
[0047] A method of preparation of the microfluidic device [300] comprising the zone devices [100, 200] is described hereinbelow.
[0048] The microfluidic device [300], comprising the Zone devices [100, 200], was fabricated on a filter paper, preferably Whatman® qualitative filter paper. The design and fabrication of the microfluidic device was done by PowerPoint drawing and heat printing respectively. HP LaserJet M1005 MFP Printer with toner hydrophobic printing material was used.
[0049] Post the designing process, the device [300] is printed onto the filter paper, wherein the printing causes depiction of the zone 1 [100], marking line [301] and zone 2 [200]. The detection zones [100, 200] are configured to hydrophobic by subjecting them to heat for about 20 minutes at 200 °C. This was performed to allow melting of the toner material to another side of the paper by penetrating down and forming the hydrophobic barrier. The heat-treated device [300] is stored in a sterile environment for further experimentation.
[0050] For Zone 1 [100], specific reagents/ enzymes are added directly onto the detection zones. For standardization of protocol, individual detection zones of size 1.2 X 1.2 mm were used. The hydrophobicity of the oven-treated paper devices was checked by using different volumes of methyl orange/methylene blue solution.
[0051] For zone 2 [200], the strip device [201] is an assembled device by consecutively connecting and partly overlapping four components, a sample pad, a conjugate pad, a test pad, and an absorbent pad. After assembly, the strip device [201] is attached in the space given at zone 2 [200]. In this embodiment, the zone 2 strip device [201] is primarily assembled before being attached to the zone 2 [200].
[0052] In another embodiment, the strip device [201] may be configured to be directly performed on the zone 2 [200].
[0053] ZONE 1:
[0054] Zone 1 [100] is configured for identifying biomarkers such as serum lipase and serum amylase.
[0055] Referring to Figure 1, the Zone 1 device [100] comprises of a sample pad [101], wherein a sample is applied to. The sample pad [101] is configured to receive a body fluid such as blood, urine, saliva as the sample. The sample pad [101] may be a porous or semi-porous pad designed to absorb and initiate the migration of the body fluid.
[0056] In an embodiment, the sample pad [101] may include anticoagulants or preservatives depending on the target body fluid.
[0057] The Zone 1 device [100] further comprises of two detection zones, i.e., sub detection zone L [102] and sub detection zone A [103]. The detection zones [102, 103] provide visual indications through colour change corresponding to the enzyme concentration. The detection zones [102, 103] indicate serum amylase and lipase presence. The Zone 1 device [100] ensures a capillary action of the body fluid in its transmission to the detection zones [102, 103] from the sample pad [101].
[0058] In a preferred embodiment, the sample pad [101] is in the form of a central circular zone (such as a 2 X 2 mm circular space) as the sample pad [101]. Emanating from the sample pad [101] are the detection zones [102, 103], preferably in the form of two smaller circles (such as 1.2 X 1.2 mm) protruding from each side of the sample pad [101].
[0059] Surface Functionalization in Zone 1 [100] is critical to enhance the sensitivity and performance of the microfluidic device [300] for enzyme detection. Surface functionalization is followed to improve the sensitivity of detection assay as the surface treatments bring certain changes/modifications in the physicochemical properties, thus surging their performances. Nanoparticles provide huge amounts of surface-active sites and high surface-to-volume ratios supporting the analyte recognition units for improvement. In this embodiment, the surface functionalisation was performed with reagents such as 0.1% Chitosan LMW: C, 1% polyethylene glycol: P, PEG - Chitosan conjugate: PC, AuNP-APTES conjugate: A-A and 0.1% Chitosan nanopowder: CNP. The Conjugates were prepared by vortexing the combination reagents in a 1:1 ratio for 15 minutes at 2400 rpm.
[0060] In this embodiment, the selection of reagents assumes significance for the reason, it improves assay results and accuracy. The reagents are added to the detection zones [102, 103] in the Zone 1 device [100]. The coatings enhance the detection of enzymes by increasing the interaction between the analyte (lipase/amylase) and the detection zones [102, 103].
[0061] In the Zone 1 device [100], the detection zones [102, 103] were individually coated with chitosan nanopowder (CNP), chitosan, polyethylene glycol (PEG), PEG-chitosan, and gold nanoparticles (AuNP) followed by the addition of p-nitro phenylbutyrate [p-NPB]. The first assay includes the detection of lipase owing to its activity of hydrolyzing p-nitrophenyl butyrate to form p-nitrophenol, yielding a yellow colour.
[0062] The treated detection zone L [102] was then subjected to different concentrations (69, 34.5, 17.25, 8.6, and 4.3) U/L of serum lipase. The action of serum lipase on the different coated detection zone [102] comprising such coatings was studied. It was observed that the detection zone L [102] with no-coating, chitosan, PEG, PEG-chitosan, and AuNP-coated zones, the colour intensity was higher as compared to CNP in 69 U/L which is considered the normal value of lipase. AuNP-coated zone displayed colour before the addition of the serum lipase standard. It was concluded that the CNP-coated zone with lower concentrations of serum lipase did not show colour, whereas, in other coated and non-coated zones, the colour development could be seen even in lower concentrations (as identified in Figure 3). Therefore, the CNP-coated zones were identified to be the optimised detection zones for identifying serum lipase in the microfluidic device.
[0063] Further, optimisation of p-NPB concentration suitable for lipase detection was also performed. Therefore, different concentrations (100, 50, 25, 20, 10, and 5) mM of p-NPB solution were used in the CNP-coated zones. All the concentrations were observed to be capable of easily distinguishing between serum lipase and normal samples with detection of lipase values between 29 U/L and 900 U/L respectively (as identified in Figure 4a). The images of the detection zones taken after the analysis were quantified using yellow colour intensity from CMYK colour space. Figure 4b identifies the graph using yellow colour intensity versus different p-NPB concentrations on CNP-coated paper devices. A marked difference was observed in all concentrations used whereas, 25 and 100 mM showed the maximum difference between normal and serum lipase samples (as identified in Figure 4b). While optimisation it was observed that the difference was more prominent within 5 minutes of sample addition, therefore, the serum lipase optimisation revealed that the CNP-coated zones, the colour of the assay zones has to be visualized within 3-5 minutes of the addition of the sample. Chitosan nanopowder is believed to have higher hypolipidemic activity when fed to rats than ordinary chitosan. The action of serum lipase on p-nitrophenyl butyrate is obstructed on the CNP-coated zone due to its hypolipidemic activity thus, in turn, delaying the release of p-nitrophenol. The lipase detection zone has 2 reference indicators (Yes: Y and No: N) with defined colours. After the assay, the developed colour on the detection zone can be matched with the Y and N wells. The CNP coating is also increasing the physical/mechanical properties of the paper-based microfluidic device.
[0064] Assay of p-NPB lipase using different dilutions of pancreatic and normal samples on the non-coated and CNP-coated paper device. A colorimetric assay was performed for lipase detection using two concentrations of p-NPB, (i) 25mM, and (ii) 100mM. Serum lipase and normal samples were diluted using MQ water into A1/R1, A2/R2, A3/R3, A4/R4, and A5/R5 (A: Patient sample; R: Normal sample). p-NPB was added once onto both non-coated and CNP-coated wells. After complete drying, different dilutions of pancreatic and normal samples were added. The colour was observed for 10 minutes and the image was recorded. The image intensity was then quantified using ImageJ.
[0065] In the Zone 1 device [100], a second assay includes the detection of serum amylase. Amylase Detection in Zone 1 utilizes a colorimetric enzymatic assay based on the Starch-Iodine-pH Indicator (SI-MR/BCG) Conjugation Assay to detect elevated levels of serum amylase.
[0066] The normal range of ??-Amylase in human beings is 30-140 U/L. In this invention, there is disclosed a method for serum amylase detection exploiting SI-MR/BCG conjugation assay. Amylase activity is detected by hydrolyzing starch, causing a colour change. When different concentrations (290, 145, 36.25, 2.26, and 0.56) U/L of amylase were added to the SI-MR/BCG conjugation coated paper zones a noticeable colour change was observed, i.e., from purplish pink to yellowish pink, and in the case of SI-MR and SI-BCG coatings, the colour change was from greenish blue to fully blue respectively.
[0067] In this, the starch-povidone complex (SI) was prepared using starch 5 %, and povidone 10% solution. To prepare SI-MR and SI-BCG solution, SI solution was mixed with the pH indicators Methyl red and BCG separately in equal ratios. Later the mixture was kept in a magnetic stirrer for 20 minutes for conjugation. As the conjugation proceeded, the colour of the solution changed from navy blue to violet colour and light green to dark green for SI-MR and SI-BCG respectively. 2.5 µL of SI-MR/BCG conjugate solution was added 4 times with subsequent drying, after each addition in different biofunctionalized paper zones (0.1% Chitosan and 0.1% CNP coated). Later, ??-Amylase (stock 290 U/L) in different dilutions (290, 145, 36.25, 2.26, and 0.56) U/L was added. Images were captured through mobile phones for analysis.
[0068] A good colour gradient was observed in both the conjugated solutions. In SI-BCG coated wells blue colour intensity was higher with higher amylase concentration which hydrolyzed the starch solution, whereas higher dilutions showed bluish-green in both the coatings (as identified in Figure 5A). In SI-MR coated wells the pink colour immediately faded and the light-yellow colour was observed in the stock whereas in higher dilutions wells pink colour was retained due to lower concentration of amylase hence the low reaction rate with SI solution (as identified in Figure 5B). In both cases good colour gradient was observed which was more prominent on the backside.
[0069] Among the 2 conjugates, SI-MR showed more prominent results (differences in colour between higher and lower dilutions) and thus was selected for further experiments with serum amylase samples. From Figure 6, it can be concluded that when the serum amylase sample with a concentration of amylase 350 U/L is added, it immediately displays a yellowish colour. Further, the serum was diluted to have a concentration of 350, 175, 87.5, 43.75, 21.8, and 10.9 U/L marked as S, S1, S2, S3, S4, and S5 respectively in the paper assay zones. It is inferred that the SI solution has a pH of about 7, when it reacts with amylase the starch is broken down into glucose and iodine directing toward a lower pH. At a pH below 6, MR shows a yellow colour. As illustrated in Figure 6, the CNP-coated wells were differentiating amylase concentration clearly, as yellow colour is seen only in the S and S1. The chitosan-coated wells to some extent, and non-coated wells failed to distinguish as it was showing a yellow colour even in the S2 with a concentration of 87.5 U/L which comes under the normal amylase level. Thus, it can be concluded that the CNP-coated wells adsorbed with SI-MR conjugate can be used to differentiate between patients based on the higher amylase values.
[0070] Figure 7 identifies the colour gradation with respect to decreasing concentration of Amylase/Lipase. In the amylase zone [103], a yellowish colour was observed in zones with higher amylase and a pinkish colour with lower amylase concentration. Whereas, in the lipase zone [102] yellowish colour was observed only in higher concentrations. Similarly for SI-BCG, blue for higher amylase and greenish blue for lower concentrations.
[0071] ZONE 2 (Colorimetric Immunoassay Zone):
[0072] Zone 2 [200] focuses on the colorimetric immunoassay for detecting Trypsinogen-2. This zone uses a Lateral Flow Immunochromatography Assay [LFIA] to provide rapid and sensitive detection. The strip device [201] was assembled and detection was done using the gold conjugation method.
[0073] The LFIA strip [201] was assembled by consecutively connecting and partly overlapping four components (as identified in Figure 8):
[0074] Sample pad [206]: The sample pad [206] receives the sample for trypsinogen detection. Preferably, it is made of glass fibre (8 X 4) mm having a thickness of 0.35 mm. The sample pad [206] is configured to receive bodily fluids such as urine, saliva, serum, and blood.
[0075] In an example embodiment, the sample pad [206] is configured to receive urine as the sample.
[0076] In another example embodiment, the sample pad [206] is configured to receive serum as the sample.
[0077] In yet another example embodiment, the sample pad [206] is configured to receive both urine/serum as the sample.
[0078] Conjugate pad [205]: It is configured to hold a conjugate, in dry and stable form until the addition of sample to the strip [201]. The conjugate pad [205] ensures rapid and clean release of the conjugates upon sample application on the sample pad [206]. Preferably, it is a Type PT-R Conjugate Release Matrix made of polyester (6 +/-2 X 4) mm.
[0079] Test pad [207]: The test pad [207] plays a crucial role in detecting trypsinogen-2 concentration. It serves as the base surface, upon which the other components are assembled, which serves as the primary surface where specific antibodies are immobilised for generating signals related to presence of trypsinogen-2. Other components, such as the sample pad [206], conjugate pad [205], and absorbent pad [202], are consecutively connected and partly overlapped onto this pad [207] during the assembly process.
[0080] The test pad [207] is a nitrocellulose membrane (of an example size such as 25 X 4) mm. Preferably, the pore size of the nitrocellulose membrane used was (12 +/-3) µm. The test pad [207] consists of a test line (T) [204] and a control line (C) [203], which are optimised for providing optimal results. The test line [204] is coated with monoclonal PRSS2 antibody and the Control line [203] is coated with anti-Rabbit IgG for assay validation.
[0081] Absorbent pad [202]: It is a layer which ensures smooth flow and absorbs excess liquid. It is Cellulose-based layer with a thickness (such as 0.80 mm) and with a strip dimension (such as 20 X 4 mm). It has a good water holding capacity with an average capacity of 52-112 µL/cm².
[0082] In an exemplary embodiment, the device of the present invention comprised of total dimensions as (55 +/- 2; X 4 +/- 1) mm.
[0083] The components of LFIA [201] were purchased from Advanced Microdevices Pvt. Ltd. and were assembled as per Figure 1. A method of assembly or preparation of the LFIA strip [201] is described hereinbelow.
[0084] The sample pad [206] was overlaid onto the conjugate pad [205], which is made of conjugate release polyester matrix to allow the speedy and clean release of conjugate on the application of the sample onto the sample pad [206]. The conjugate pad [205] along with the sample pad [206] was placed at the top surface of the test pad [207], at its one end. At the other end of the test pad [207], the absorbent pad [202] is overlaid. In a preferred embodiment, the nitrocellulose membrane comprised a pore size of 15 µm.
[0085] Gold nanoparticles (AuNP) were synthesized by the Turkevich method with minor modifications. Lab-synthesized AuNP was evaluated by ultraviolet-visible (UV/Vis) spectrophotometry at a wavelength range of 400 to 700 nm and absorbance maxima was recorded at 530 nm. The antibody-conjugated nanoparticles showed a shift towards higher wavelengths. There was a small red shift in the max absorbance spectrum between the AuNP-RIgG conjugate and bare AuNP. Whereas, max absorbance spectrum change was observed in the case of AuNP-mAb.
[0086] To check whether all the detector antibodies are efficiently bound to AuNP, 5µl of 2M NaCl was added to 5µl of prepared conjugation solutions, and no black colour due to AuNP aggregation was observed endorsing effective conjugation. When all the antibodies are not bound by AuNP, free AuNP experiences aggregation as NaCl decreases the charge repulsion amid gold nanoparticles thus causing its aggregation.
[0087] For capture antibody (Ab) concentration optimization in the control line [203], anti-Rabbit IgG in different concentrations (0, 0.1, 0.25, 0.5, 1, 1.5, and 2) mg/mL were added onto the test pad [207] and marked respectively as 0, 0.1, 0.25, 0.5, 1, 1.5, and 2. After adding buffer in the sample zone [206], the control line [203] colour intensity was analysed. No reaction was observed in the test strip marked as 0. The test strips marked as 0.1, 0.25, and 0.5 disclosed reactions in the control zone, in the form of a dot with not much difference in the colour intensity. The reaction in the test strips marked as 1, 1.5, and 2 were observed in the form of an arc, with bright colour intensity (as identified in Figure 9). The reaction was observed within 2 minutes. Therefore, it was concluded that the optimal sample concentration required for identifying antibody is in the range of 1-2 mg/mL.
[0088] Figure 10 identifies a lateral flow assay strip [201] displaying colour intensity at the control line [203] after the addition of buffer. The final parameters for the control line [203] were optimized as capture Ab concentration at 1-2 mg/mL, conjugate preparation at 250 µL of AuNP, conjugate pad [205] and test pad [206] incubation time as 1 hour at 37?, and the control line [203] to be kept at a distance of 12 -15 mm from the conjugate pad [205].
[0089] The preparation of the AuNP– PRSS2 Monoclonal Antibody/anti-Rabbit IgG Conjugate for Test line [204] and Control line [203], is described hereinbelow.
[0090] The choice of using either polyclonal or monoclonal antibodies was based on the affinity of Tryp-2 protein and the intensity of the test line [204] colour. Optimization of test line assay was tried using both polyclonal and monoclonal antibodies as a capture antibody. The conjugate solution (mAb PRSS2-AuNP) was used as a detector molecule for detecting trypsinogen -2 biomarker (Trypsin-2 (PRSS2) Recombinant protein) along with capture antibodies (pAB and mAb) immobilized onto the test line [204] of the test pad [207]. The choice of using both polyclonal and monoclonal antibodies as a capture antibody is to show that both can be used as a capture antibody and have an affinity towards the trypsinogen- 2 biomarker. This is useful in the identification of trypsinogen-2 biomarker. Referring to Figure 11, it can be observed that the reaction could be seen in the test strip [201] as the solution passed it. A complete coloured line can be observed in both the test lines [203, 204] after 2 minutes and a clear colour development was observed within 5 minutes. The colour intensity in the test strip [201] where 1 µg/ml was added was higher as compared to the strip [201] where 0.5 µg/ml was added in both cases. To develop a complete lateral flow assay, both control and test lines [203, 204] are added together on the test pad [207]. The amount of conjugate solution adsorbed onto the conjugate pad [206] directly affects the assay result i.e. the test and control band colour intensity. Therefore, optimization of the volume of the conjugate solution (both test and control conjugate solution) has been performed. The assay was carried out as above and only the amount of conjugate solution added was changed. The Test: Control (T: C) conjugate solution was added in the portion of 4:4, 4:3, 6:4, and 6:6 (with 2.5 µL of test and conjugate added each time/portion). Figure 12 displays the colour intensity of the LFIA strip [201] after 5 minutes of addition of the sample solution. It can be observed that the maximum intensity where the test bands are distinctly visible in both test and control zones was in the T: C; 6:4 added test strip. In the 4:4, and 4:3 strips the reaction can be seen but very faintly. Whereas, 6:6 failed to show any reaction in the test zone. Thus, it can be concluded that 4 times control conjugate and 6 times test conjugate when added shows efficient test lines in both the zones.
[0091] A method for conjugation of AuNP–Rabbit IgG for reaction at the Control line [203], is described hereinbelow.
[0092] Lab-prepared AuNP, pH 8.0 +/- 0.4 (500 & 250) µL were taken in two Eppendorf tubes. To each of these, 5µL of 100 µg /Rabbit IgG was added. The tubes were kept in a vortex mixer at 700 rpm for 5 mins. Onto these 5µL of 10% BSA solution was added and kept at RT for 5 mins. Both the tubes were then centrifuged for 10 mins at 12,000 rpm. After several steps of resuspension and centrifugation, the final conjugate mix was reconstituted in conjugate buffer mix (0.01 M PBS with BSA, sucrose, trehalose, and NaN3) and stored at 4 °C. The optical density (OD) was optimized based on dilution using a UV–visible spectrophotometer before coating onto the conjugate pad. Both (500 & 250) µL lab-prepared AuNP-conjugate was used for control strip analysis.
[0093] A method for conjugation of AuNP– PRSS2 Monoclonal Antibody for reaction at the Test line, is described hereinbelow.
[0094] Lab-prepared AuNP pH (8.0 +/- 0.4), 250 µL were taken in the Eppendorf tube. To this, 10 µL of 100 µg/ml of PRSS2 Monoclonal Antibody was added. The tube was incubated at RT for 30 minutes. Onto this 50µL of 10% BSA in 20mM PBS was added and kept at RT for 15 mins. It was then then centrifuged at 12,000 rpm for 20 mins. The supernatant was discarded and the AuNP-Ab conjugate was resuspended in 500 µL of 10mM PBS. The solution was again centrifuged at 9000 rpm for 20 mins. The final conjugate was reconstituted in 50µL of conjugate buffer mix (10mM PBS along with 1% BSA, 5% Sucrose, 5% Trehalose, and 0.1% sodium azide) after discarding the supernatant and stored at 4 °C until use.
[0095] The OD was optimized based on dilution and characterization was carried out using a UV–visible spectrophotometer before coating onto the conjugate pad.
[0096] The Final Optimization of Control Line [203] is described hereinbelow.
[0097] An assay strip of (25 X 4) mm was prepared by attaching a sample pad, a conjugate pad by removing adhesive at one end, and at another end an absorbent pad was attached. To the conjugate pad 2.5 µL of the conjugate solution, Rabbit IgG- AuNP conjugate as mentioned above was added 6 times followed by incubation at 1 hour at 37? for drying. Conjugate pads were pretreated with 2 % blocking solution and dried at 37? for 30 minutes before adding conjugate solution.
[0098] The control line [203] was kept at a distance of 15mm from the top of the strip. In the control line, a semicircle was marked with a sharp object, and 0.6 µL of 2mg/mL Anti-Rabbit IgG along with 2% Methanol 6 times was added, thus making the total volume 3.6 µL. The strip with capture antibody was then dried at 37? for 1 hour. A blocking solution was added below and above the control line very precisely without touching the control mark. After drying, a conjugate pad was assembled in the strip, and added 20µL 1X Phosphate buffer for the flow to occur. Images were taken and recorded.
[0099] The standardisation of parameters for test line is described hereinbelow.
[0100] Mouse anti-human PRSS2 Monoclonal Antibody (Catalog Number MBS7111088, MyBioSource) Antibody was used to prepare detector antibody along with lab-prepared AuNP as discussed above. Briefly, 500 µL of AuNP and 10 µL of 100µg/ml MAb for 30 minutes at RT. This was followed by the addition of 100µL of 10% BSA in 20 mM PBS and again incubating at RT for another 15 minutes. After this, the solution is centrifuged at 12,000 rpm for 20 mins. The AuNP-Ab conjugate is resuspended in 1 ml of 10 mM PBS after discarding the supernatant. The solution is again centrifuged at 9000 rpm for 20 min. The final conjugate is reconstituted in 100µL of 10 mM PBS containing 1% BSA, 5% Sucrose, and 0.1% sodium azide and is stored at 4? until use.
[0101] The LFIA components consisted of a sample pad (8 X 5) mm, a conjugate pad (3 X 5) mm, a test pad (25 X 5) mm. and an absorbent pad (20 X 5) mm. Two strips of the above-mentioned dimensions were prepared and assembled. The test line was marked in dot form with a distance of 5mm from the conjugate pad. Onto this, 0.6µL of 200 µg/ml of Rabbit anti-Human PRSS2 Polyclonal Antibody was added in the test zone 6 times, before the next addition the test line was dried completely. After this, a blocking solution was added below and above the test zone. Conjugate pads were first coated with 2.5µL of blocking solution followed by fully dipping in 15µL of conjugate solution for 15 minutes. After the addition of the reagents, both the conjugate pads and test strips were kept in the incubator for 1 hour 45 minutes at 37?. Finally, the conjugate pad and absorbent pad were assembled onto the strip before analysis above and below the test line respectively.
[0102] For analysis and as a sample solution, Trypsin-2 (PRSS2) Recombinant protein (Catalog Number MBS958920, MyBioSource) was used. 40µL of sample solution (1 and 0.5) µg/ml was added to the sample pad of each strip and allowed to get absorbed for 45 seconds followed by the addition of 40µL of running buffer (10mM PBS) onto the sample pad. The strips were then analyzed for reaction in the test strip and the image was taken and recorded.
[0103] A Test line assay using Monoclonal Antibody as a capture antibody is described hereinbelow.
[0104] Two test strips were assembled as discussed in the above sections and marked as T1000 and T500. The test line was marked 5mm from the conjugate pad. Conjugate pads were first coated with 2.5µL of blocking solution followed by fully dipping in 10 µL of conjugate solution for 15 minutes followed by incubation at 37? for 30 mins. 3.6 µL of 100 µg/ml mAb was added and kept in the UV cross-linker at 0.165 Joule/cm2 for 2 minutes followed by an incubation at 37? for 2 hours.
[0105] Conjugate and sample pads were assembled onto the strip and analysis was done using 40 µLof sample solution (Human Trypsinogen-2) in a conc. 1000 ng/mL, and 500 ng/mL. Later, 40µL of running buffer was added to the sample pads. The result was recorded by taking images.
[0106] To develop a complete lateral flow assay for both control and test lines [203, 204] are marked together on the test pad [207]. Conjugate solutions for both the control and the test line were prepared as discussed above. To the conjugate pad [205] 5µL of blocking solution was added and dried at room temperature, followed by the addition of AuNP– PRSS2 mAb (6 times) and Rabbit IgG- AuNP (4 times). Each addition required the pad [206] should be completely dry before further addition for better absorption. After complete addition, the conjugate pad [206] was incubated at RT for 15 mins followed by 37?/30 mins incubation. Subsequently, six strips of the previously mentioned dimensions were prepared, assembled, and marked as 0, 62.5, 125, 250, 500, and 1000 depicting sample concentrations (0, 62.5, 125, 250, 500, and 1000) ng/mL. The test & control zones were marked with the help of a micro tip end at a distance of 7 mm, and 12mm respectively from the top of the test membrane. Above and below these marked zones, blocking solution (1- 2) µL and allowed to dry at RT followed by the addition of the same volume of double distilled water without touching the marked zones. After complete drying, 3.6 µL (0.6µL at one time) each of 100µg/ml PRSS2 mAb and 2mg/mL Anti-Rabbit IgG in 2% methanol was added respectively at test and control zones. The test pads were kept in the UV Cross-linker (365 nm), at 0.165 Joule/cm2 for 2 minutes followed by incubation at 37? for 2 hours. For analysis, 40µL of Trypsin-2 (PRSS2) Recombinant protein sample solution in 6 different concentrations (0, 62.5, 125, 250, 500, and 1000) ng/mL were added onto each strip and allowed to get absorbed for 45 seconds followed by the addition of 40µL of running buffer (10mM PBS) onto the sample pad. The strips were then analysed for reaction in the test strip and the image was taken and recorded.
[0107] In Figure 13, the efficacy of the developed assay protocol is demonstrated for detecting the Trypsinogen -2 protein efficiently even at a lower concentration. Trypsinogen -2 protein was taken in different concentrations (0, 31.25, 62.5, 125, 250, 500, and 1000) ng/mL and added to the sample pads. When the solution is added to the sample pad, the Trypsinogen -2 protein migrates to the conjugate pad and reacts with the monoclonal PRSS2 Ab which is specific to the Trypsinogen -2 protein and due to specific antigen-antibody reaction, an Ag-Ab-AuNP complex is formed. The complexes Tyr-2-PRSS2-AuNP/AuNP-RIgG migrate to the test pad.
[0108] Tyr-2-PRSS2-AuNP forms a sandwich antigen-antibody complex with another monoclonal Ab specific against another epitope of Tyr-2 and gets trapped there, whereas AuNP-RIgG migrates towards the control zone and forms Ag-Ab complex with anti-RIgG immobilized there. The sandwich reaction in the test zone allows the gold-conjugated antibodies to accumulate at that point thus forming of red/purple colour. The colour intensity in the test line is proportional to antigen concentration in the sample. The higher the concentration of antigen more intense the test line (as identified in Figure 13). The colour band in the control zone is due to the accumulation of gold nanoparticles at the antigen-antibody reaction site. Trypsinogen-2 concentration is strongly elevated in persons with acute pancreatitis and is considered a very potential marker.
[0109] This assay device [201] presents the advantages of enzyme detection as well as lateral flow assay in a single platform. Several assay parameters, including the detection card design, AuNP volume, distance of test and control zones, and number of enzymes immobilized for detection, were standardized to boost the detection sensitivity of this single Enzymatic/Immunochromatography platform. The visual detection limit for immunoassay is 57 -55 ng/mL and can be detected in 2 minutes, whereas, the lipase and amylase detection zones can differentiate between samples within 1 minute.
[0110] In order to evaluate the efficacy of the zone device [200], real-time blood samples (5 normal and 5 acute pancreatitis) were collected from MNR Hospital and Dr. Gopal Krishna, Gastroenterologist, in Sangareddy, Telangana. All samples were tested for trypsinogen-2 levels using the ELISA method before testing using the zone device [200]. Figures 14 – 16 illustrate the tests conducted on such samples and the observed results.
[0111] Different concentrations of human Trypsinogen-2 were detected in a microtiter ELISA plate using the Human PRSS2 (Protease, Serine 2) ELISA Kit (MBS8801064, HSN Code: 38229090, MyBiosources) according to the manufacturer's protocol. Five normal samples and five patient samples were selected for the study. In brief, wells were designated as (S1-S5) for five normal control samples, a standard sample, and a blank. One hundred µL of the respective samples were added to the corresponding marked wells. In the standard and blank marked wells, 100 µL of stock standard solution and sample diluent buffer were added, respectively. The wells were wrapped in aluminium foil and incubated at 37? for 80 minutes. After thorough washing with wash buffer and drying, 100 µL of 1X Biotinylated Ab working solution was added to each well and incubated at 37? for 50 minutes. This was followed by washing, drying, and the addition of 1X Streptavidin-HRP working solution, which incubated for 50 minutes at 37?. The wells were finally dried, and then the solution was added in the dark. Colour development was achieved by adding 90 µL of TMB substrate solution containing substrate-dye buffer to each well and incubating at 37? for 15 minutes, followed by the addition of 50 µL of stop solution (5% H2SO4) to halt colour development. Absorbance was recorded at 450 nm using an ELISA reader (Thermo Scientific, Multiscan FC), and a standard curve was generated. The same procedure mentioned above was followed for the patient samples as well.
[0112] In case of normal samples, the absorbance values of all samples were below the absorbance values for the standard: 50 ng/mL.
[0113] In contrast, the recorded values for patient samples were as follows: (S1: 14, S2: 23, S3: 125, S4: 33, and S5: 74) ng/mL. The lower values in patient samples may be due to the timing of sample collection, as trypsinogen-2 levels peak within a few hours of abdominal pain in acute pancreatitis before returning to normal within 3 days. The test device is intended for point-of-care diagnosis during episodes of abdominal pain.
[0114] In patient samples, the absorbance values of the standard were 0.4087 which denotes 50 ng/mL of Tryp-2; absorbance values of patients’ samples S1-S5 were recorded as 0.1469, 0.1887, 1.0262, 0.3263, and 0.6110, respectively, which corresponds to the Trypsinogen-2 concentration of (14, 23, 125, 33, and 74) ng/mL. Only 2 samples showed values higher than 50 ng/mL, which is also seen with a faint line on the LFIA.
[0115] Test strips were prepared as discussed and marked for both control and test lines on the test pad. Testing was done by adding 40µL of serum sample solution onto each strip and allowing it to get absorbed for 45 seconds, followed by the addition of 40µL of running buffer (10 mM PBS) onto the sample pad. The strips were then analyzed for reaction in the test zones, and the image was taken and recorded. In both normal and patient samples testing, along with real samples, positive (1000 ng/mL) and negative (0 ng/mL) control solutions, i.e., recombinant trypsinogen-2 protein, were also investigated. A sample showing trypsinogen-2 concentrations of 125 ng/mL was tested independently 3 days to validate the assay performance.
[0116] From the figure 14, it is observed that control lines appeared in all seven test strips, whereas the test line was visible only in the positive control with trypsinogen-2 concentration of 1000 ng/mL. Whereas no test line was visible in the strips with trypsinogen-2 concentrations lower than 50 ng/mL. The LOD for the developed strip is 50 ng/mL; hence, trypsinogen-2 proteins below this level cannot be detected.
[0117] The concentration of trypsinogen-2 in patients samples as calculated by ELISA were (S1: 14, S2: 23, S3: 125, S4: 33, and S5: 74) ng/mL, similar results were observed in the strips with test line appearing in 2: 500, 3: Positive control, 6: Sample 3, and 8: Sample 5 having trypsinogen-2 concentration of 1000, 500, 125, and 74 ng/mL respectively (Figure 15). No test line was observed in test strips 1, S1, S2, and S4, having trypsinogen-2 concentrations of 0, 14, 23, and 33 ng/mL, respectively. The intensity of the test line also decreases with decreasing concentration of protein.
[0118] A sample showing trypsinogen-2 concentrations of 125 ng/mL was tested independently 3 days to validate the assay performance. It was observed that in all 3 days, a good test line was visible in the test line along with a clear line in the control zone (Figure 16).
[0119] The consistent, strong signal in the samples having trypsinogen-2 concentrations higher than 50 ng/mL, confirmed successful assay performance.
[0120] The above-mentioned description illustrates and depicts various embodiments of the present invention. However, it will be appreciated that numerous changes and modifications are likely to occur as per user requirements, and it is intended in the appended claims to cover all these changes and modifications which fall within the true spirit and scope of the present invention. ,CLAIMS:WE CLAIM:
1. A multiplexed microfluidic assay device [300], comprising:
a first zone/Zone 1 device [100] configured for colorimetric enzymatic assay to detect serum lipase and serum amylase; and
a second zone/Zone 2 device [200] configured for colorimetric immunoassay to detect trypsinogen-2 biomarker using a lateral flow immunochromatography assay.
2. The device as claimed in claim 1, wherein the device [300] is separable into two zone devices, i.e., Zone 1 [100] and Zone 2 [200] at the marker [301].
3. The device as claimed in claim 1, wherein the zone devices [100, 200] are configured to receive a sample in the nature of bodily fluid selected from saliva, blood, serum, and urine.
4. The device as claimed in claim 1, wherein the zone devices [100, 200] provide visual indicators in the nature of colour gradation, as an indicator of presence of lipase, amylase, and trypsinogen-2.
5. The device as claimed in claim 1, wherein the zone 1 device [100] comprises:
a sample pad [101] for receiving the sample; and
sub-detection zones [102, 103], wherein the first sub-detection zone is for lipase and the second sub-detection zone is for amylase.
6. The device as claimed in claim 1, wherein Zone 1 device [100] is surface functionalised with coatings selected from chitosan nanopowder, polyethylene glycol, PEG-chitosan, and gold nanoparticles.
7. The device as claimed in claim 1, wherein the reagent for the first sub-detection zone [102] is p-nitrophenyl butyrate (p-NPB).
8. The device as claimed in claim 1, wherein the first sub-detection zone [102] is configured to detect serum lipase by hydrolyzing p-nitrophenyl butyrate to form p-nitrophenol, yielding a yellow colour,
9. The device as claimed in claim 1, wherein the second sub-detection zone [103] is coated with starch-iodine-pH indicator conjugates (SI-MR/BCG) as the substrate.
10. The device as claimed in claim 1, wherein the second sub-detection zone [103] is configured to detect serum amylase by hydrolyzing starch, causing a colour change based on pH indicators.
11. The device as claimed in claim 1, wherein the sample pad [101] may optionally include anticoagulants or preservatives to facilitate sample migration and analysis.
12. The device as claimed in claim 1, wherein the sub-detection zones [102, 103] are hydrophobic.
13. The device as claimed in claim 1, wherein the detection limit for trypsinogen-2 is 50 ng/mL, enabling rapid detection within 2 minutes.
14. The device as claimed in claim 1, wherein the zone 2 device [200] comprises a test strip [201] including components such as a sample pad [206], conjugate pad [205], test pad [207], and absorbent pad [203].
15. The device as claimed in claim 1, wherein the sample pad [206], conjugate pad [205], and absorbent pad [203] are assembled onto the test pad [207].
16. The device as claimed in claim 1, wherein the test pad [207] is marked with test line [204] and a control line [203].
17. The device as claimed in claim 1, wherein the test pad [207] is a nitrocellulose membrane.
18. The device as claimed in claim 1, wherein the absorbent pad [203] is a cellulose layer.
19. The device as claimed in claim 1, wherein the optimal sample concentration or capture Ab concentration required for zone 2 device [200] is 1-2 mg/mL.
20. The device as claimed in claim 1, wherein the conjugate is linked to AuNP.
21. The device as claimed in claim 1, wherein the test line [204] is optimally placed at 7 mm from the tip end.
22. The device as claimed in claim 1, wherein the control line [203] is optimally placed at 12 – 15 mm from the conjugate pad [205].
23. A method for preparing a multiplexed microfluidic assay device [300], the said method comprises:
fabricating detection zones [100, 200] on a qualitative filter paper using hydrophobic toner material;
wherein the fabrication involves printing the detection zones [100, 200] on the filter paper;
subjecting the fabricated paper to heat treatment to induce hydrophobicity; and
storing the heat-treated paper/assay device [300] in sterile environment.
24. The method as claimed in 23, wherein the detection zones [100, 200] are separated by a marking line [301]; and wherein the detection zones [100, 200] are separable at the marking line [301].
25. The method as claimed in 23, wherein the heat treatment is performed at a temperature of 200°C for 20 minutes.
26. A method of preparing Zone 1 device [100] of a multiplexed microfluidic assay device [300], the said method comprising surface functionalization of sub-detection zones [102, 103] with biofunctionalized materials selected from Chitosan LMW, Polyethylene Glycol, PEG-Chitosan conjugate, gold nanoparticles (AuNP) and Chitosan nanopowder (CNP).
27. The method as claimed in 26, wherein the reagents are vortexed in a 1:1 ratio for 15 minutes at 2400 rpm to prepare conjugates.
28. The method as claimed in 26, wherein the first sub-detection zone [102] is coated with p-nitrophenyl butyrate (p-NPB) as the substrate.
29. The method as claimed in 26, wherein the second sub-detection zone [103] is coated with starch-iodine-pH indicator conjugates (SI-MR/BCG) as the substrate.
30. The method as claimed in 26, wherein the starch-povidone complex (SI) is prepared using 5% starch and 10% povidone solution, followed by conjugation with methyl red (MR) or bromo cresol green (BCG).
31. The method as claimed in 26, wherein the sub-detection zones [102, 103] are optimized to indicate colour gradation within 3-5 minutes of sample addition.
32. A method for preparing Zone 2 device [200] of a multiplexed microfluidic assay device [300], the said method comprises:
assembling a lateral flow immunochromatography assay (LFIA) strip [201] by consecutively connecting and overlapping four components such as a sample pad [206], conjugate pad [205], test pad [207], and absorbent pad [203].
33. The method as claimed in 32, wherein the test pad [207] forms the base of the strip [201]; and wherein the sample pad [206], the conjugate pad [205] and the absorbent pad [203] are assembled on the test pad [207].
34. The method as claimed in 32, wherein the sample pad [206] is made of glass fibre.
35. The method as claimed in 32, wherein the conjugate pad [205] is a polyester matrix.
36. The method as claimed in 32, wherein the test pad [207] is a nitrocellulose membrane.
37. The method as claimed in 32, wherein the test pad [207] comprises a pore size in the range of 12 ± 3 µm.
38. The method as claimed in 32, wherein the optimal concentration of capture antibody for the control line is 1-2 mg/mL.
39. The method as claimed in 32, further comprising optimization of the conjugate solution volume for the test and control lines, with a ratio in the range of 4:4 to 6:6.
40. The method as claimed in 32, wherein the test line is prepared using monoclonal PRSS2 antibody conjugated with AuNP, enabling antigen-antibody reactions specific to trypsinogen-2.