Development of a New Microtiter Plate Format for Clinically Relevant

Jan 17, 2012 - A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization...
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Development of a New Microtiter Plate Format for Clinically Relevant Assays Elena V. Piletska,*,† Stanislav S. Piletsky,† Michael J. Whitcombe,† Iva Chianella,† and Sergey A. Piletsky† †

Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, U.K. ABSTRACT: A new format for the microtiter plate-based assays was proposed. The novelty involves the use of diskshaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The “mix and match” approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

The microtiter plate (or microplate) assay is a very popular format for clinical and environmental analyses. It allows fast and reproducible testing of 96 samples in common laboratory plates and 1536 samples in high throughput screening plates. Microtiter plates are generally used for chemical and biological experiments such as detection and monitoring of chemical or biological reactions, cell growth, and toxicity tests and can be applied in combinatorial chemistry.1−5 It is impossible to overestimate the range of target compounds, which are routinely quantified in microtiter plates worldwide and how much personnel time and cost it saves while increasing the precision and dramatically minimizing the volumes of the measured samples. Over the years intensive optimization work has been done on the immobilization of biological, chemical compounds and polymers onto the walls of microtiter plates.6,7 However, the relatively low surface area of microtiter wells allows only small quantities of reagents (e.g., antibodies) to be immobilized onto their surfaces. Some modification and further improvement of the capabilities of the microtiter plate format resulted from the introduction of inserts, microcolumns, or raised pads.8−10 However, the introduced materials tend to obstruct the pathway to light which is required for the measurement of analytes in the microplate wells and therefore make it necessary to transfer the samples to different plates for measurement. Having mentioned numerous advantages of the microtiter plate format, it is necessary to stress that most assay protocols involve multiple additions of reagents which require time, skill, and patience on the part of scientists and technicians and so are often a source of errors. The idea of using disk-shaped inserts (Figure 1) was mainly aimed at minimizing the required manipulation to two easy © 2012 American Chemical Society

Figure 1. The picture of the disks made of glass microfiber GF/F.

steps: the addition of the analyte and the measurement of a color change produced by an enzymatic or chemical reaction. The latter arises from the interaction of the analyte with reagents preadsorbed onto the inserts. The disk format would also increase the effective surface area of the microtiter plate wells and allow the unobstructed measurement to be performed in the same plate. The components that are used in the microtiter plate-based assays typically include biological species (e.g., enzymes, proteins, antibodies and nucleic acids), buffers, reaction substrates, dyes, and other reagents. The important fact is Received: December 8, 2011 Accepted: January 16, 2012 Published: January 17, 2012 2038

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Bicinchonic Acid (BCA) Protein Assay. Protein measurements were performed using a modified BCA Protein Assay.11 According to the manufacturer’s protocol12 BCA Protein Assay reagent A contains sodium carbonate, sodium bicarbonate, BCA, and sodium tartrate in 0.2 N sodium hydroxide; reagent B contains 4% cupric sulfate. 100 GF/F disks were soaked in reagent A of the BCA kit and 100 disks in reagent B, which was diluted 50 times with water. The disks were then dried on the filter paper and assembled into the microtiter plate: one disk with reagent A and one disk with reagent B per well, placed one on top of each other. In order to assess the performance of the disk-based BCA assay, 100 μL of BSA standard solutions (0.015−1 mg mL−1) was added to each well in three replicates of each concentration and incubated for 30 min at 37 °C accordingly to the manufacturer’s protocol. The absorbance was measured using a Dynex microtiter plate reader at 600 nm. All measurements were made in triplicate. Measurement of pH. The possibility to use universal indicator preadsorbed on the glass microfiber disks for the measurement of pH in the microtiter plate format was demonstrated. 100 GF/F disks were soaked in 10 mL of 2% (w/v) solution of universal indicator (Fisher, UK) for 5 min. The excess of liquid was removed using filter paper, and disks were then dried for 10 min under vacuum in a desiccator. GF/F disks containing universal indicator were placed in the wells of the microtiter plate. 100 μL of 0.1 M sodium phosphate buffers (PB, pH 4.5−8.5) and 0.1 M sodium borate buffer, pH 9.0, were added to each well containing a disk with universal indicator and incubated for 5 min. The response of the universal indicator toward different pHs was measured using a microtiter plate reader (Dynex, UK) at 600 nm. All measurements were made in triplicate. Quantification of Glucose Using Glucose Oxidase and Universal Indicator Preadsorbed on the Glass Microfiber Disks. 100 GF/F disks were soaked in 10 mL of a 0.6% (w/v) solution of universal indicator containing 70 mM PB, pH 7.0, for 5 min. 100 GF/F disks were soaked in 10 mL of GOx (1 mg mL−1) for 5 min. The excess liquid was removed using filter paper, and the disks then were dried for 10 min under vacuum in a desiccator. One of each type of disk was placed in each of the wells of the microtiter plate, one on the top of the other. Standard glucose solutions (100 μL) in the concentration range 1−100 mM were added to the microplate wells and incubated for 30 min. The response was measured using a microtiter plate reader at 650 nm. All measurements were made in triplicate. Quantification of Urea Using Urease Reaction and Universal Indicator Preadsorbed on the Glass Microfiber Disks. 100 GF/F disks were soaked in 10 mL of a 0.6% (w/v) solution of universal indicator (Fisher, UK) containing 70 mM PB, pH 7.0, for 5 min. 100 GF/F disks were soaked in 10 mL of a solution of urease (1 mg mL−1) for 5 min. Excess liquid was removed using filter paper, and the disks were dried for 10 min under vacuum in a desiccator. One of each type of disk was placed in the wells of the microtiter plate, one on the top of each other. Standard urea solutions (100 μL) in the concentration range 1−25 mM were added to the wells, and the plate was incubated for 60 min at room temperature. The response was measured using a microtiter plate reader at 650 nm. All measurements were made in triplicate. Assay for Antioxidants. The protocol described by Steinberg et al.13 was modified for the disk assays format. Twelve mg of radical DPPH was dissolved in 9 mL of THF.

that due to their reactive nature, in standard microtiter platebased assays some of the reagents cannot be mixed in advance; therefore, they are usually added one by one just before (or after) the addition of the sample. In the proposed assay format all reactive components are preadsorbed onto the microfiber disks (one or several), dried, assembled in the right combination in the microplate, and stored at the required temperatures until the plate is used. This approach is especially invaluable for assays which require the addition of more than two components, like enzymatic reactions, where enzymes and substrate are added separately. The advantage of using the diskshaped inserts is the opportunity to measure the samples in the same plate through the central opening without any additional liquid-handling, therefore preserving the best features of the microtiter format. One microtiter plate could contain a selection of the preassembled disks for the measurements of a range of analytes, which are required for the diagnosis of a particular medical condition. The combination of the multianalyte microtiter plates with bioinformatics software installed onto laboratory microtiter plate reader could allow medical practitioners to obtain a comprehensive assessment of the patient’s condition in a short time. In order to demonstrate the proof of principal for this new approach, we selected several clinically relevant reactions which could be measured in the microtiter plate format. These include the following: · BCA Protein Assay for measuring protein concentration; · pH measurements using universal indicator; · enzymatic reactions with glucose oxidase to measure concentration of glucose; · enzymatic reaction with urease to measure concentration of urea; and · 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay for antioxidants.

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MATERIALS AND METHODS Materials. Universal indicator (UI, 2% w/v) was purchased from Fisher (Loughborough, UK). Urease from Jack bean (Type III) (urea amidohydrolase, EC 3.5.1.5) and glucose oxidase (GOx, EC 1.1.3.4) from Aspergillus niger, 2,2-diphenyl1-picrylhydrazyl (DPPH), urea, D-glucose, gallic acid, sodium hydroxide, sodium phosphate dibasic, and sodium phosphate monobasic were purchased from Sigma-Aldrich (Gillingham, UK). HPLC-grade water was purified using Milli-Q apparatus (Millipore, UK). Bicinchonic (BCA) Protein Assay kit was produced by Pierce (Thermo Scientific, UK). Disk Preparation. The disks with external diameter 7 mm and internal diameter 3 mm were cut from 4 different materials: filter paper (Whatman No. 3), MF membrane filters containing nitrocellulose (Millipore), glass microfiber GF/D, and glass microfiber GF/F (both from Whatman) using 25 W Fenix Laser Marker carbon dioxide laser marking system fitted with 80-mm lens (Synrad Inc., Mukileto, USA). The dimension of the disks was selected to fit into the wells of standard microtiter plates with flat bottoms. In order to immobilize the reagents (buffers, UI or solutions of the enzymes) 100 disks were soaked in 10 mL of a solution of the required reagent for 5 min, the excess of liquid was adsorbed with a generic filter paper, and disks were then dried for 10 min under vacuum in a desiccator and stored in closed glass vials until use. 2039

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The results of the disk-based BCA Protein Assay showed that there is a correlation between the protein concentration and assay response over a wide concentration range 0.015−1 mg mL−1 (Figure 2).

100 glass microfiber GF/F disks were incubated in the solution of DPPH for 5 min, then removed, dried on a filter paper, and packed into microtiter plates. Aliquots (100 μL) of solutions of gallic acid in PBS, pH 7.4, in the concentration range 0.015−1 mM were added to each well. The plate was incubated for 30 min at room temperature, and the absorbance of each well was measured using a microtiter plate reader at 450 nm. All measurements were made in triplicate.

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RESULTS AND DISCUSSION The Preparation of Disks and Their Performance in BCA Protein Assay. Several different materials were tested for disks preparation, including filter paper, nitrocellulose, and two different glass microfiber filters. The disks were cut using a computer-controlled CO2 laser. Disks were treated with soluble reagents as described in Materials and Methods and tested in BCA Protein Assay. BCA Protein Assay is a standard method which is used for the determination of protein concentration. It relies on the reaction between peptide bonds of proteins, Cu2+, and two molecules of BCA leading to the formation of purple-colored BCA-Cu1+ product (eq 1)

Protein (peptide bonds) + Cu 2 + → tetradentate‐Cu1 + complex Cu1 + + 2BCA → purple BCA‐Cu1 + complex

(1)

Figure 2. The readout of the microtiter plate with reagents of the BCA reaction preadsorbed on the microfiber disks in the presence of the BSA in the concentration range 0.015−1 mg mL−1 at wavelength 600 nm (a); a photograph of the plate (b).

The macromolecular structure of the protein, the number of peptide bonds, and the presence of three amino acids (cysteine, tryprophan, and tyrosine) are reported to be responsible for color formation with BCA.14 Since the solutions of reagents used in the assay are not stable upon mixing, their application in a disk-based format offers serious advantages over standard microtiter plate-based assays. Thus, both reagents A and B, dried on two disks and placed in the same microplate well, would react with each other only following addition of an analyte solution and prior to this can be stored for long periods (up to 3 months) without any change in their performance. Although all materials tested here showed promising performance in the BCA Protein Assay, the best results were obtained with GF/F glass filters from Whatman (Figure 1). This type of filter had superior mechanical strength and did not swell in water or in organic solvents. In addition it demonstrated the highest response with 2 mg mL−1 of BSA in the BCA reaction (Table 1). The high sensor response was most likely caused by the higher adsorption ability of porous GF/F filters.

pH Measurement. The variation in pH values is an important diagnostic parameter which could indicate the presence or degree of progress of an illness, making the measurement of pH very important.15 In addition to blood, urine, and cerebrospinal fluids, which are commonly tested for biochemical analysis, there are other biological liquids, such as pleural, peritoneal, and pericardial fluids, whose pH values are indicative of the effectiveness of treatments and can be used as a diagnostics tool.16 One of the most traditional methods for measuring the pH of urine samples is litmus-impregnated dipsticks. Although this method is very easy and affordable, it does not provide sufficient precision of the measurement. The microtiter plate format perfectly suits the measurement of pH because it allows simultaneous analysis of a large number of small-volume samples. The introduction of the disks impregnated with universal indicator can be considered as a further improvement of pH measurements. The proposed pH assay relies only on one disk with a mixture of preadsorbed dyes (universal indicator). In fact, the universal indicator consists of several pH-sensitive dyes such as phenolphthalein, methyl red, bromothymol blue, and thymol blue, specifically designed for measurements throughout a wide pH range. The disks with dyes could be stored for long periods (up to 3 months) without any loss in their performance. When brought into contact with buffer, the preadsorbed dyes were rapidly dissolved showing a stable adsorption at 650 nm, which could be directly correlated with the pH value of the solution (Figure 3). Glucose Oxidase Assay. The disks impregnated with universal indicator are suitable for monitoring of enzymatic reactions which are accompanied by pH changes. In order to

Table 1. BCA Response to 2 mg mL−1 of BSA with Reagents Preadsorbed on the Disks Made from Different Materialsa absorbance, au

a

material

H2O

BSA

Δ, ads

comments

Whatman filter No. 3 MF/nitrocellulose Whatman glass GF/D Whatman glass GF/F

0.24 0.13 0.44 0.21

0.39 0.27 0.45 0.87

0.15 0.14 0.01 0.66

low density, floats too flexible fluffy well-defined disks

The measurement was performed at λ = 600 nm.

Typically one disk of GF/F glass microfiber with surface area 20 mm2 adsorbed 7.0 ± 0.1 mg of reagent solution. 2040

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Figure 3. The readout of the microtiter plate with universal indicator preadsorbed on the glass microfiber disks in the presence of different buffers in the pH range 5.5−8.0 at wavelength 650 nm (a); a photograph of the plate (b).

Figure 4. The readout of the microtiter plate with reagents of the GOx reaction preadsorbed on microfiber disks in the presence of the glucose in the concentration range 1−100 mM measured at 650 nm (a); a photograph of the plate (b).

prove this concept, we used reactions catalyzed by glucose oxidase (GOx) and urease. GOx catalyzes the oxidation of β-Dglucose to β-D-gluconolactone,17 which is then hydrolyzed to gluconic acid; therefore, GOx reaction is accompanied by a shift in pH toward the acidic range (eq 2)

catalyzes the hydrolysis of urea to ammonia and carbon dioxide according to the reaction (eq 3)20

(NH2)2 CO + H2O → CO2 + 2NH3

Glucose + O2 + 2H+

In the disk-based assay two sets of disks with preadsorbed urease and universal indicator were prepared as described in Materials and Methods and packed in microplate wells on the top of each other. Upon addition of urea solution the components were rapidly dissolved and the reaction began. The assay shows a direct correlation between the presence of urea in the physiological range of concentrations, 1−25 mM, and the color change of UI (Figure 5). The linear concentration range of the disk-based assay for the determination of urea is similar to the commercial urea enzymatic assay kit which is based on the calorimetric detection of urea in the microtiter plate format and measuring the urea in liquids in the range between 40 and 1200 ppm (0.7−20 mM).21 The standard deviations in the detection of analytes measured in disk-based assays were under 5% which is acceptable for the practical application. The urease is a stable enzyme. The disks with immobilized urease could be stored for more than 4 months at +4 °C without loss of performance. Based on the examples presented here it is possible to conclude that universal indicator is a sensitive and effective marker for the quantitative measurement of reactions which result in changes of pH to occur. In principle it is possible to combine disks impregnated with universal indicator with other enzymes which would allow multiple tests to be conveniently performed at the same wavelength (e.g., 650 nm) to measure many analytes simultaneously. Assay for Antioxidants. An assay was developed for measuring antioxidants, such as gallic acid, using the stable lipophilic chromogenic radical DPPH. One set of disks with

→ Gluconolactone + H2O2 → Gluconic acid

(3)

(2)

For the glucose oxidase assay, two sets of disks with adsorbed GOx and universal indicator were prepared as described in Materials and Methods and packed into microplate wells. Upon addition of glucose solution, the components were rapidly dissolved and the reaction proceeded. The results showed that there was a direct correlation between the concentration of glucose in the range 1−100 mM and the response of GOx with universal indicator impregnated on the microfiber disks (Figure 4). In comparison with traditional methods for the measurement of glucose, which require two enzymes (GOx and peroxidase),18,19 the method with universal indicator and GOx has clear advantages: it is more convenient and less prone to variations in enzyme activity and the prepackaged assay could be stored at +4 °C for long periods (more than 6 months) without loss of performance. The linearity of the responses achieved in our work is similar to those achieved in liquid assays for glucose detection.19 The standard deviations in the detection of analytes measured in triplicates were under 5% which is acceptable for the practical application. Urease assay. Another enzymatic assay that was tested in combination with universal indicator was the urease assay. Similarly to the glucose oxidase reaction above, the urease assay is accompanied by pH changes, the difference being that the pH changes are shifting toward the basic range. Urease 2041

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radical molecule. DHHP-treated disks were stored in the dark glass vial at room temperature for more than a month without any loss of the activity toward antioxidants. As compared with standard optical sensor based on the polymer films described by Steinberg et al.13 the advantage of the disk assay format lies in easy preparation, smaller standard deviations, and the possibility to apply it to the rapid measurement of large numbers of samples.

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CONCLUSIONS

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AUTHOR INFORMATION

A new format for microtiter plate assays was presented and illustrated by several clinically relevant examples. The described disk-based approach is generic and could be applied to various assays and analytes. One of the advantages of this format is that one microplate could house several assays which could be preassembled for a specific clinical or diagnostic need. Of the materials tested, the disks prepared from glass microfiber filters showed the best performance as the support material, both for various microtiter plate-based assays as well as for assays which are not compatible with polystyrene microtiter plates. Among the advantages of the new format is the possibility to perform a range of assays rapidly and without the need to handle multiple liquid reagents. The liquid-handling protocol can be reduced to a single addition of sample to the microtiter plate, prepackaged with disks containing the necessary reagents.

Figure 5. The readout of the microtiter plate with reagents of the urease reaction preadsorbed on microfiber disks in the presence of the urea in the range between 1 and 25 mM at wavelength 650 nm (a); a photograph of the same plate (b).

preadsorbed DPPH was prepared as described in Materials and Methods and packed into microplate wells. The addition of gallic acid (which is used as a standard in the measurement of antioxidant activity) caused an intense color change following 30 min incubation (Figure 6).

Corresponding Author

*Phone: +44(0)1234 758325. E-mail: [email protected]. uk.

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REFERENCES

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Figure 6. The readout of the microtiter plate with reagents of the antioxidant assay preadsorbed on microfiber disks in the presence of gallic acid in the range 0.015 to 0.5 mM at the wavelength 450 nm (a); a photograph of the same plate (b).

The obtained calibration for gallic acid was linear in the range between 0.015 to 0.5 mM (Figure 6). DHHP is a stable free2042

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