Effect of cationic polyelectrolytes on the performance of paper diagnostics for blood typing

Effect of cationic polyelectrolytes on the performance of paper diagnostics for blood typing

Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal h...

3MB Sizes 0 Downloads 0 Views

Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Effect of cationic polyelectrolytes on the performance of paper diagnostics for blood typing Heather McLiesh, Scot Sharman, Gil Garnier ∗ BioPRIA, BioProcessing Research Institute of Australia, Department of Chemical Engineering, Monash University, Clayton, VIC 3800, Australia

a r t i c l e

i n f o

Article history: Received 8 December 2014 Received in revised form 23 May 2015 Accepted 29 May 2015 Available online 10 June 2015 Keywords: Bioactive paper Antibody Red blood cells Retention Blood typing Polyelectrolyte

a b s t r a c t We investigated the effect that two common types of cationic polyelectrolytes used in papermaking might have on the performance of paper diagnostics using blood typing as an example. The results were analyzed in terms of red blood cells (RBC) retention and antibody–antigen specificity. Two questions were addressed: (1) can poly(amido-amine) epichlorohydrin (PAE) typically used for paper wet strength affect the diagnostic performance? (2) can high molecular weight cationic polyacrylamide (CPAM) employed as retention aid enhance or affect the selectivity and sensitivity of paper diagnostics? A series of paper varying in type of fibers and drying process were constructed with PAE and tested for blood typing performance. Residual PAE has no significant effect on blood typing paper diagnostics under normal conditions. Positives are unaffected with PAE, while negatives lose slight sharpness as some RBCs are unselectively retained. CPAM, the most common retention aid, can also be used to retain cells and biomolecules on paper. Paper towel was treated with CPAM solutions varying in polymer concentration and charge density and tested for blood typing. We found that CPAM dried on paper can retain RBC. CPAM affects the negative tests by retaining non-specifically individual RBC on fibers. RBC retention increases non-linearly with the CPAM charge density and concentration. As expected, wet CPAM retain RBCs at concentrations higher than 0.1 wt%. As paper diagnostics are becoming a reality, more realistic papers than the Whatman filter paper will be engineered. This study provides guidance on how best use the required polymeric wet-strength and retention agents. Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.

1. Introduction Bioactive paper has emerged as a new generation of products combining a biomolecule as specific agent with a cellulose fiber/surface used as functional substrate. Pelton [1] provided a comprehensive review on bioactive paper. A rapidly expanding application of bioactive paper is for biomedical applications; these were reviewed by a few research groups [2–7]. A paper-based diagnostic consists of a cellulose fiber based substrate on which an analytical system is deposited. This paper diagnostic is usually wetted with a biofluid to detect and quantify a biomolecule such as an enzyme, antibody, a protein or a hormone – and a response is read. As paper is used wet, and samples are often very valuable, a wet-strength agent and retention aid will be required for mass producing paper diagnostics. However, little is known on the effect

∗ Corresponding author. Tel.: +61 3 9905 9180. E-mail address: [email protected] (G. Garnier). http://dx.doi.org/10.1016/j.colsurfb.2015.05.049 0927-7765/Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.

such polymeric agents – typically cationic polyelectrolytes – might have on the behavior of the paper diagnostic. A new generation of low cost and easy to use paper diagnostics has recently been developed for blood typing [8–13]. The concept relies on specific antibody–antigen interactions to selectively agglutinate red blood cells (RBC) on paper, to separate the agglutinated RBC (positive) from the non-agglutinated RBC (negative), to directly communicate results; the paper test can also be retained as a document. Red blood cells (RBC) agglutinated from antibody-specific haemagglutination reactions transport differently on paper compared to non-agglutinated blood [8]. The basis of immunohaematology used in blood typing diagnostics is nicely reviewed elsewhere [14–16]. The non-agglutinated RBCs can wick paper and transport by capillary flow; agglutinated RBC cannot transport on paper and become retained, forming a visual indicator of a positive reaction. This concept laid the foundation for developing paper-based blood typing devices. Subsequently, Al-Tamimi et al. [9] developed a paper-based assay for rapid blood typing.

190

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

The new method involved fixation of RBCs on paper treated with blood typing antisera and an elution step. RBCs in contact with the specific antibody agglutinated and fixed onto the paper while non-agglutinated RBCs were eluted through capillary action. For a sensitive and reliable test, it is critical that a positive response yields strong agglutination and retention of the red blood cells (RBC) on paper upon elution, therefore providing a clear visual signal; for a negative test, all RBC need to be completely washed off the paper, leaving it pristine white. While filter paper (Whatman mostly) has been the preferred substrate for bioactive paper [17–22], this material cannot be translated into product development and commercial process. Filter paper is not only too expensive but in most cases shows poor performance especially for blood typing [10]. Tissue and light weight paper substrates of controlled structures and porosity have been shown to be more effective [8–11]. However, two types of polymeric additives are required to develop colloids retention and strength of these lightweight non-woven fibrous composites typically used wet: retention aids and wet strength agents. These polymeric additives, such as cationic polyacrylamide (CPAM) and polyethyleneimine (PEI) retention aids are typically cationic; wet-strength agents, such as poly(amido-amine) epichlorohydrin (PAE) are able to bind covalently with cellulose when heated at 100 ◦ C. The chemical structures of these two polyelectrolytes are shown in Fig. 1. A critical issue is whether or not these polymers can affect the retention or desorption of the red cells and antibody used on the paper diagnostics. Of particular concerns are the false positive, in which red blood cells are indiscriminately retained on paper, or false negative, for which positive and normally retainable cells are eluded [2]. Paper diagnostics for blood typing rely on two mechanisms: elution/chromatography in the plane of paper and filtration through paper [11]. We previously observed that filter paper with surface treatments behaved differently than those without which affected typing clarity of the positive and negative tests [10]. We also reported that potentiators often used in the formulation of antibody solution could contribute to false positive [11]. Potentiators such as dextran and polysaccharides are sometimes added to the formulation of the commercial antibodies to enhance the RBC coagulation when using antibodies with low avidity resulting in weak blood group reactions [15]. However, potentiators and retention aids can also be needed to improve the strength and size of RBC aggregates, especially in the case of weak or secondary blood groups. This study has two objectives. The first is to elucidate whether wet-strength agents such as poly(amido-amine) epichlorohydrin (PAE) and common papermaking retention polymers, such as cationic polyacrylamides (CPAMs) can affect blood typing analysis and retain RBC or antibodies on paper. A requirement of the antibody physisorbed on paper is its ability to desorb and diffuse within the blood droplet, therefore agglutinating the biospecific RBC. The second objective is to quantify the effect of cationic polyelectrolyte charge density and concentration on the retention of RBC and antibody and to analyze how these affect the sensitivity of positive and negative tests. Three studies are performed. In the first, PAE is adsorbed on pulp fibers prior to papermaking and dried. The polymer effect on antibody, RBC retention and diagnostic performance is then evaluated. In the second study, a series of CPAM at various charge densities is investigated as retention aid for antibody on paper and tested for positive and negative bio-specific test with RBC. The effect of CPAM charge density on antibody and RBC bridging ability and the consequences are analyzed. In the third study, the retention ability of wet CPAM adsorbed on paper is analyzed in the context of paper based blood typing analysis. It is the objective of this study to investigate cationic polyelectrolytes to improve paper based blood typing analysis performance.

2. Experimentals 2.1. Materials The cationic polyacrylamides (CPAMs) were kindly supplied by AQUA + TECH (Switzerland) and used as received. These are copolymers of uncharged acrylamide with cationic dimethylaminoethylacrylate methyl chloride having 5%, 10%, 20%, 30%, 50% of charge monomer and MW 13 MDa. The 80% charge CPAM had MW 8 MDa as defined by the manufacturer. Poly(amido-amine) epichlorohydrin (PAE) was supplied from Nopco, Australia. The standard Professional Kleenex paper towel from Kimberly–Clark, Australia was used as substrate for most experiments. It has a basis weight of 26.4 g/m2 and is a trilayer sheet. A Filter paper grade 1845 having a pore size 25 ␮m and a basis weight of 70 g/m2 was purchased from Filtech and used as comparison. Three EDTA stabilized blood samples, one of each group A, B and O, stored at 4 ◦ C, were supplied by Australian Red Cross Blood Service (Sydney) and were used between 10 and 14 days post collection. Blood typing antisera used was FFMU (For Further Manufacture Use) anti-A and anti-B purchased from Alba Bioscience, Edinburgh, United Kingdom. The washing solution used was PBS (Phosphate Buffered Solution) prepared with MilliQ water and PBS tablets supplied by Sigma–Aldrich. 2.2. Methods 2.2.1. PAE paper preparation Paper handsheets were prepared according to the Australian/New Zealand Standard Method 203s. Basically the dry pulp was thoroughly wetted by soaking in deionized water for about 12 h. The pulp was transferred to a disintegrator (Model MKIIIC, Messmer Instruments Ltd.), diluted to 2 L with deionized water and disintegrated for 75 000 propeller revolutions. If required, the PAE solution was added to the pulp slurry prior to handsheet forming and stirred for 5 min. The pH of the pulp slurry mixture was not adjusted and the value was about 5. The addition quantity of PAE (20 mg/g fiber) was based on oven dry grammage of 60 g/m2 . After manual couching and wet-pressing at 0.4 MPa for about 15 s, the sheets were either air dried 24 h at 23 ◦ C and 50% relative humidity (RH) or cured in a SEMMAR Auto Dryer Type MR-3 at 112 ◦ C for 10 min, in order to activate covalent bonds between the PAE and cellulose. Each paper type was cut into 40 individual 3 cm × 3 cm squares. Anti-A and anti-B were each dispensed on to 20 squares of all the prepared papers. Positive test used A cells and B cells, respectively, while O cells was the negative test with 10 replicates of each using the same blood testing method as below. 2.2.2. CPAM treated papers CPAM solutions were prepared as follows. Six differently charged CPAMs were dissolved at 1 g/L in deionized water and vigorously mixed for 48 h, followed by two serial one in five dilutions, in deionised water and mixed for 24 h after each dilution. This resulted in three concentrations: 1 g/L, 0.2 g/L and 0.04 g/L, for the CPAM treated paper testing. Likewise, two serial one in ten dilutions of 30% charged CPAM were made for the wet CPAM testing resulting in 1 g/L, 0.1 g/L and 0.01 g/L solutions with a minimum 24 h mixing for each step. Paper toweling was prepared by drawing a dozen 6 cm × 6 cm squares onto 72 sheets. Each CPAM solution (100 ␮L) was dispensed onto the middle of all 12 squares on 4 sheets of the prepared paper towel, ensuring no contact between paper towel and bench. The paper towel with CPAM was subsequently dried in a SEMMAR Auto Dryer Type MR-3 (112 ◦ C for 10 min) prior to testing. Once dry, the sheets of paper towel were all tested in the same manner. In addition, an extra 24 sheets of paper towel were prepared with a dozen 6 cm × 6 cm squares. CPAM 30% charge solution

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

191

Fig. 1. Chemical structure of (A) poly(amido-amine) epichlorohydrin (PAE) and (B) cationic polyacrylamide (CPAM). PAE can develop a covalent bond with the hydroxyl of cellulose upon heating. The CPAM used is a copolymer of uncharged acrylamide with cationic dimethylaminoethylacrylate methyl chloride.

(100 ␮L) was dispensed onto 8 sheets paper towel per dilution: 0.01 g/L, 0.1 g/L and 1 g/L. Half were drum dried (112 ◦ C for 10 min), and the other half were tested when still wet. Both testing of CPAM treated papers and wet CPAM on the paper towel were examined in the same manner. On each piece of paper there were 4 columns of 3 squares. Blood was added to each column as follows: (1) directly onto the CPAM treated paper, (2) after the addition of PBS, (3 and 4) after anti-A was dispensed. Group A blood was used in the first 3 columns and group O blood in the final column as a negative control. 2.2.3. Blood testing method Antiserum or PBS (10 ␮L) was dispensed into the squares on the paper towel, followed by 3 ␮L of mixed EDTA blood. Group A blood was used for positive test and group O blood for the negative test. The blood spots were left to incubate in the laboratory for a minimum of 30 s. Following incubation, the test paper was placed over blotting paper and washed 3 times with 100 ␮L PBS drop by drop. The test was moved over to a clean blotting paper for each wash. The tests were left to dry overnight at room temperature then scanned on an EPSON 2450 scanner. The intensities of the scanned images were read by imageJ program. The results were collated with average and standard deviation values calculated. 3. Results and discussion 3.1. Effect of wet-strength agent on RBC retention and diagnostic specificity The first study investigated whether a typical cationic polyelectrolyte used as wet-strength in the fabrication of paper and tissue can retain antibodies and red blood cells on paper under normal conditions, therefore contributing to false positives through systematic non-selective adsorption. Poly(amido-amine) epichlorohydrin (PAE) is a common wet-strength agent typically adsorbed from solution onto the pulp fibers at 2–10 kg/T fibers (Fig. 1A). Paper handsheets were constructed from two types of fibers: eucalyptus (hardwood) and spruce (softwood) fibers, each with the wet-end addition of 20 mg PAE/g fiber. Half of these handsheets were air dried (at 50% relative humidity, 23 ◦ C), while the

other half was dried by heating at 112 ◦ C. These papers were then tested with antibody and RBC using the standard flow-through methodology. Fig. 2 schematically illustrates the experimental procedure. A filter paper selected to filtrate only RBC aggregates was also tested as comparison. The pictures of a typical positive and negative response for each of the 9 papers tested are shown in Fig. 3. A few observations are of interest. First, all papers showed a marked difference between the positive and negative test. Positives display a relatively well-defined red dot of high color intensity; a halo is sometimes visible at the boundary. Negatives do not form dots and show less clear boundaries; trace and stain left by RBC are visible. Second, all papers made with PAE as additives are yellowish. Third, there are no obvious differences between papers made with softwood and hardwood fibers, or air-dried versus heated. The color intensity of blood dots for positive and negative was compared for the 9 types of papers. The positive and negative tests for A and B type blood are illustrated in Fig. 4. The average and standard deviation of 10 tests are represented for each case. There are a few commonalities. First, the intensity of positives is always significantly higher (usually double) than the negatives, confirming the concept for the test. Second, the type of RBC antigen (A versus B) does not affect results. The intensity of positive tests for RBC type A is slightly higher than for RBC type B, while negatives are identical; anti-A has a greater avidity than anti-B. The group B agglutinates are more likely to break up during the washing process. Third, the intensity of negatives for RBC is slightly higher on softwood fibers made paper than on hardwood paper (7 out of 8 tests). This represents slightly higher retention by filtration of the cells on softwood paper. Fourth, filter paper specifically selected for the large pore sizes 25 ␮m (greater than RBC, 8 ␮m) and a comparable weight to the papers made, provides strong intensity positives, but also retain some individual RBC as shown by the relatively high intensity of the negative test. This filter paper is preferable over previously filter papers tested for blood typing [10]. Fifth, the negative of all papers containing PAE is higher than without, while the positives are unaffected. This suggests that PAE can retain some individual RBC by non-specific electrostatic mechanism, which contributes to decreasing the sensitivity of the test. Sixth, papers cured at high temperature have higher intensities negatives than those dried at room temperature. This was seen in both papers with PAE and

192

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

Fig. 2. Schematic representation illustrating the protocol of blood type testing using different papers made with and without PAE.

without; this probably indicates a slight change of fibers or paper morphology upon thermal treatment. The area of the stain formed by the blood droplet reacting with the antibody previously deposited on paper is compared for the

9 types of papers with anti-A and anti-B (Fig. 5). There are a few interesting observations. First, the size of blood drop is smaller on hardwood made paper than on paper from softwood fibers. This is consistent with the bigger pores on paper made with the

Fig. 3. Effect of the type of fiber and the drying process on the clarity of response of paper blood typing diagnostics made with PAE as wet strength agent. Positive and negative blood typing diagnostics on hardwood fiber paper and softwood fiber paper. Anti-A positive test: group A cells, negative test: group O cells. Anti-B positive test: group B cells, negative test: group O cells. Filter paper of similar basis weight is shown for comparison.

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

193

Fig. 4. Effect on PAE, type of fiber and drying process on the intensity of blood spot on paper. HW = hardwood, SW = softwood, DD = drum dried, AD = air dried, PAE = PAE added. (a) Anti-A with group A and O cells and (b) anti-B with group B and O cells. Standard deviation, shown by error bars, n = 10.

bigger fibers of softwood able to wick a bigger quantity of liquid before drying occurs. Second, positives of blood droplets reacting with the more avid A antibody have a smaller area than positives with B antibody. Third, the area of negative dots stain is higher than that of the positive and not as clear, as illustrated with the higher standard deviations. Fourth, the addition of PAE increased the spot size (14 of 16 pairs). Fifth, spot size of the negative tests is slightly larger on drum dried than the air-dried papers (14 out of 16 pairs). These observations confirm the minimal effect PAE has on blood typing paper diagnostics. Positives are basically unaffected with PAE, while negatives lose a slight sharpness as some RBCs are unselectively retained. When considering a diagnostic device, the intensity value gives greater clarity than the spot size to distinguish the positives from the negatives, especially on hardwood with no PAE (Figs. 4 and 5). The poly(amido-amine) epicholohydrin (PAE) polymer chain contains cationic monomers (amine) and reactive units

(azetidinium); the former group retains PAE on cellulose, the second cross-links cellulose for wet-strength development upon heating. PAE can affect the activity of antibodies immobilized on paper in 2 ways: (1) unspecific retention and (2) deactivation of the antibody. As expected, the residual charge of reacted PAE is insufficient to retain RBC by electrostatic interaction; this is confirmed by the very similar negative tests for paper with and without PAE. This suggests that unspecific RBC retention is not an issue at low concentration of PAE (less than 20 mg/g). None of the papers tested exhibited perfect negative: all showed some low level of residual red color intensity upon washing, indicating the retention of RBCs. These RBCs are very likely retained as individual cells and by mechanical action, either by entrapment on individual fibers or within the pores of paper. The filter paper exhibits negatives tests similar in color intensity to those of the other papers (thickness/basis weight are similar). Since filter paper has an average pore diameter (25 ␮m) over 3 times the diameter

194

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

Fig. 5. Effect on PAE, type of fiber and drying process on the blood spot area (in cm2 ) on paper. HW = hardwood, SW = softwood, DD = drum dried, AD = air dried, PAE = PAE added. (a) Anti-A with group A and O cells and (b) anti-B with group B and O cells. Standard deviation, shown by error bars, n = 10.

of the RBC (7 ␮m; height = 2 ␮m), which are highly flexible and deformable, it is very likely that individual RBCs retain within the grooves, roughness or fibrillation characteristics of cellulosic fibers. Our results corroborate the findings of Wang et al. [23] who studied the effect of PAE wet-strength resin and the type of paper on the activity of IgM antibodies adsorbed on paper. Pelton reported that PAE does not have a dramatic positive or negative effect on the activity of paper-supported antibodies. PAE at 10–20 mg/g paper improved antibody performance, whereas higher PAE concentrations decrease the bioactive paper efficiency [23]. PAE is typically used at 1–10 mg/g paper for wet-strength applications. PAE deactivation of antibody is therefore not an issue at the concentrations of interest. 3.2. Effect of CPAM on RBC retention and diagnostic specificity High molecular weight cationic polyacrylamides (CPAMs) are often used as retention aids in papermaking. CPAM is typically added at 0.4–2 kg/T to the fiber suspension prior to promotes the retention of fines and filler. High molecular weight polyelectrolytes retain colloids onto fibers by 2 mechanisms: bridging and charge reversal. The effect dry CPAM can have on the performance of paper

diagnostic for blood typing is investigated. This study quantifies the effect that charge density and concentration of a high molecular weight CPAM (13 MDa) can have on antibody and RBC retention on paper. The concern is that the residual charge left from a CPAM adsorbed and dried on paper can affect the performance of paper blood typing diagnostics. Two questions were investigated: (i) can CPAM treated paper retains red blood cells? (ii) Can CPAM adsorbed on paper be used to increase the clarity or sensitivity of tests? Increased sensitivity can be achieved by increasing the intensity of antibody specific positive, while the antibody non-specific RBC (negative) remains unaffected. Paper was treated with CPAM solutions varying in polymer charge density and concentration, then dried following the standard technique (112 ◦ C, 10 min). The first column was left dry, the second had PBS spotted on it, and antibody anti-A was spotted on the final two columns: A type RBCs were then deposited onto the first three columns (positive) and O type blood on the last column (negative), incubated 30 s and washed with 3× 100 ␮L PBS as before (Fig. 6). The intensity of the resulting RBC dot was measured (Fig. 7). The typical results are presented in triplicate in Fig. 6 for CPAM of low (20%) and high (80%) charge density polymer at 3 polymer concentrations (0.04 g/L, 0.2 g/L, 1 g/L), for specific (A) and non-specific (O) RBC. All positives tests (A cells on antibody anti-A) present a

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

195

Fig. 6. Picture of blood retention on CPAM treated paper – antisera and RBCs were deposited onto the CPAM spot for (A) 20% charge density CPAM and (B) 80% charge density CPAM polymer solution at 0.04 g/L, 0.2 g/L and 1 g/L. (Column 1) Group A cells added directly to the paper, (2) group A cells added to 10 ␮L PBS, (3) group A cells added to 10 ␮L anti-A and (4) group O cells added to anti-A.

clear dot. All negative (O RBC on antibody anti-A) are much more feinted than the positives and very distinct – at one exception. The exception is with CPAM of the highest charge (80%) and concentration (1 g/L) – offering maximum retention potential – whereby the negative appears positive – albeit of lower intensity. Reproducibility is excellent as shown by the similarity between the 3 replicates. This test was repeated for solutions of high molecular CPAM (13 MDa) differing in concentration and charge density. The average and standard deviation of 12 positives and negative replicates

Fig. 7. Effect of the CPAM charge density and solution concentration on the RBC retention measured as intensity. (A) Anti-A + A cells (positive) 1 g/L CPAM. (B) AntiA + O cells (negative) 1 g/L CPAM. (C) Anti-A + O cells 0.2 g/L CPAM. (D) Anti-A + O cells 0.04 g/L CPAM. (E) Anti-A + O cells no CPAM. Standard deviation, shown by error bars, n = 12.

is shown in Fig. 7. For positives, high intensity dots are formed by the RBC. Not only the intensity of the signal is independent of CPAM charge, but CPAM does not increase the intensity of the positive. This might be due to the sufficiently high RBC and antibody concentration and the saturating of color intensity past a high threshold. Experiments were conducted at high RBC and antibody concentration; lower concentrations can be explored to increase contrast of positives. However, using CPAM affects the negative tests – here group O RBC on antibody anti-A (also group A cells on the PBS with no antibodies and added directly onto the paper). The effect of CPAM on retaining unspecific RBC starts at a concentration of 0.2 g/L CPAM. This effect becomes very strong for more concentrated CPAM solutions (1 g/L), and CPAM charge density is an important variable. RBC retention increases non-linearly with CPAM charge. CPAM charge density becomes important on negative tests using non-specific RBC above 30%; at 80% charge, the intensity value of the negative is almost reaching that of the positive. It is interesting to note that the negatives on the lower dilutions of CPAM on the paper towel have a consistently lower intensity reading than all the negatives on the papers used in the PAE study. This is likely due to the cumulative effect of the paper towel having a lower thickness, and a lower concentration of PAE (typically 2–10 mg/g) than the papers made in this study (20 mg/g). The CPAM of charge density 10% and 50% (13 MDa) form a monolayer on microfibrillated cellulose (MFC) at 8 and 5 mg/g, respectively [24]; Charge neutrality (zeta potential,  = 0), were achieved at the same CPAM/MFC ratios (MFC  = −26 mV) [24]. MFC has a surface area of 35 m2 /g versus 1 m2 /g for most papers; the maximum CPAM surface coverage is 0.22 mg/m2 and 0.14 mg/m2

196

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

Fig. 8. Comparison of CPAM 30% charge wet and dry. (Column 1) A cells added directly onto paper; (2) A cells added to 10 ␮L anti-A; (4) O cells added to 10 ␮L anti-A.

for the CPAM of 10% and 50% charge density, respectively [24]. With 100 ␮L CPAM solutions droplets forming circles of diameter of 6 cm giving an area of 28 cm2 and considering the above CPAM full surface coverages, it can be calculated with the paper towel (26.4 g/m2 ), that full CPAM monolayer coverage are achieved at concentration of 0.16 g/L and 0.10 g/L for the 10% and 50% charge density CPAM. These CPAM concentrations correspond to the levels at which the red blood cells are seen to retain significantly on paper (Fig. 9).

3.3. CPAM as biomolecule and cell retention aid CPAM is a common retention aid used in the paper industry to retain process colloids on fibers. In water treatment, CPAM is used for coagulating dissolved solids and colloids. In both instances, CPAM is employed wet and the process relies on the bridging ability of the hydrated random polymer coils. In this study, the effect of a high molecular weight CPAM (13 MDa) of constant charge density (30%) on the clarity of paper blood typing was investigated both wet and heat-dried. Paper was pretreated with CPAM solutions of different concentrations, and blood typing was measured for positive and negative tests. Fig. 8 presents how the paper diagnostics clarity for positive and negative tests is affected by CPAM solution concentration and drying process. The intensity of the blood spots as a function of CPAM concentration is presented in Fig. 9 for the various tests. The tests on the wet CPAM show an enhanced intensity of blood typing reaction at the higher dilutions over the heat-dried CPAM. While the CPAM is wet it is able to capture the RBC with higher efficiency. However at the lower concentrations, wet CPAM is unable to capture the RBCs which are able to flow through the paper easily, leaving the paper without a stain from the cells; this contrasts from the dried CPAM.

The retention of RBC by CPAM molecules adsorbed on wet paper was certainly expected. Blood is a stable colloid suspension with moderately charged RBC having a zeta potential of −13.7 mV [25]. The high molecular weight cationic polyelectrolyte (13 MDa) retains RBC on paper by a combination of bridging and charge reversal. More interesting is the retention of RBC by CPAM heat dried and cured on paper which can be achieved by 2 mechanisms. The first is by electrostatic interaction between the residual cationic charge of the adsorbed CPAM and the negatively charged RBC. The second possible mechanism is the rehydration and relaxation of CPAM extending from paper to form a polymer layer into the liquid blood droplet; the complete CPAM desorption from paper to blood is very unlikely (all bonds must desorb at the same time).

Fig. 9. Effect of CPAM concentration on the RBC retention on paper towel measured as intensity. Retention of A and O cells with anti-A on drum dried CPAM 30% charge and wet CPAM using a logarithmic scale. Standard deviation, shown by error bars, n = 12.

H. McLiesh et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 189–197

The first phenomenon (residual charge) is certainly a major contributor to RBC retention as proven by the increase in retention upon polymer charge density (Fig. 7). Less clear is the likelihood of CPAM to rehydrate and form a bridging layer, of thickness higher than the Debye length of RBC in blood (nm) during the time frame of the test (min). The maximum layer thickness would be that of CPAM coils adsorbed from solution onto a cellulose surface. This layer was 4.3 nm as measured by neutron reflectometry at the cellulose–liquid interface (CPAM 40% charge, MW 13 MDa) [26]; for comparison the calculated radius of gyration (rg ) is 18.9 nm [26], and an IgM has a diameter of a 30 nm [27].

197

might improve results at low concentration of antibody as preliminary results suggest. However, CPAM affects the negative tests by retaining unspecifically individual RBC on fibers. RBC retention increases non-linearly with CPAM charge density and concentration. As expected, wet CPAM retain all RBC at concentrations higher than 0.1 wt%. As paper diagnostics become a reality, more realistic papers than the model Whatman filter paper will be engineered. This study provides guidance on how best use the required polymeric wetstrength and retention agents. Acknowledgements

4. Conclusion This study investigated the effect that two common types of cationic polyelectrolytes used in papermaking might have on the performance of paper diagnostics. Blood typing paper diagnostic was chosen as application for the many types of specific and unspecific interactions involved as an antibody interacts with red blood cells containing a series of selective antigens in their membranes. The results were analyzed in terms of red blood cells (RBC) retention and antibody–antigen specificity. Two questions were addressed: (1) can poly(amido-amine) epichlorohydrin (PAE) used as paper wet strength affect biodiagnostic performance? (2) Can high molecular weight cationic polyacrylamide (CPAM) employed as retention aid enhance or affect the selectivity and sensitivity of paper diagnostics? A series of paper varying in type of fibers (hardwood, softwood) and drying process were constructed with PAE added at 20 mg/g to the fiber suspension, and tested for blood typing performance. PAE is the most common wet-strength agent and is widely used in tissues and papermaking. Residual PAE has no significant effect on blood typing paper diagnostics under normal conditions (less than 20 mg/g). Positives are basically unaffected with PAE, while negatives lose slight sharpness as some RBCs are unselectively retained on paper by PAE. Similarly, the other variables affecting paper structure such as the type of fiber (fine hardwood fibers versus coarse softwood fibers), and the drying temperature, have minimal effect on the results, with the grammage of paper (thickness, porosity) having the greatest. CPAM is the most common retention aid used in papermaking. For bioactive paper, it could also be used to retain cells and functional biomolecules: enzymes, antibodies, proteins. Paper towel was treated with a series of CPAM solutions varying in polymer concentration and charge density. These CPAM treated papers were sequentially spotted with antibody solutions and blood (specific and non-specific) on the CPAM dried or still wet. We found that CPAM heat-dried on paper can retain RBC. In this study, CPAM did not increase the retention – or color intensity – formed by RBC in positive tests, probably due to the high concentration of antibody and cells used, the intensity threshold for the test had been reached. Only high concentration of cells and antibody were tested; CPAM

This work was funded by the Australian Research Council Grant (LP110200973) and Haemokinesis. CPAM were kindly supplied by Aqua + Tech Specialties, Switzerland. EDTA blood samples were supplied by ARCBS (Sydney). References [1] R. Pelton, Trends Anal. Chem. 28 (2009) 925. [2] W.L. Then, G. Garnier, Rev. Anal. Chem. 32 (2013) 269. [3] W.L. Then, G. Garnier, in: S. L’Anson (Ed.), 15th Fundamental Research Symposium, Vol. 2, Cambridge, 2013, p. 541. [4] C. Rozand, Eur. J. Clin. Micro Infect. Dis. 33 (2014) 147. [5] J. Hu, S. Wang, L. Wang, F. Li, B. Pingguan-Murphy, T.J. Lu, F. Xu, Biosens. Bioelectron. 54 (2014) 585. [6] A.K. Yetisen, M.S. Akram, C.R. Lowe, Lab Chip 13 (2013) 2210. [7] E.J. Maxwell, A.D. Mazzeo, G.M. Whitesides, MRS Bull. 38 (2013) 309. [8] M.S. Khan, G. Thouas, G. Whyte, W. Shen, G. Garnier, Anal. Chem. 82 (2010) 4158. [9] M. Al-Tamimi, W. Shen, Z. Rania, T. Huy, G. Garnier, Anal. Chem. 84 (2012) 1661. [10] J. Su, M. Al-Tamimi, G. Garnier, Cellulose 19 (2012) 1749. [11] W.L. Then, M. Li, H. McLiesh, W. Shen, G. Garnier, Vox Sang 108 (2) (2015) 186. [12] M.S. Li, J.F. Tian, M. Al-Tamimi, W. Shen, Angew. Chem. 51 (2012) 5497. [13] P. Jarujamrus, J. Tian, X. Li, A. Siripinyanond, J. Shiowatana, W. Shen, Analyst 137 (2012) 2205. [14] G. Daniels, I. Bromilow, Essential Guide to Blood Groups, Wiley-Blackwell, Chichester, West Sussex, UK, 2010. [15] D.M. Harmening, Modern Blood Banking and Transfusion Practices, F. A. Davis Company, Philadelphia, PA, 2012. [16] Y. Lapierre, D. Rigal, J. Adam, D. Josef, F. Meyer, S. Greber, C. Drot, Transfusion 30 (1990) 109. [17] A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Angew. Chem. Int. Edit. 46 (2007) 1318. [18] E.M. Fenton, M.R. Mascarenas, G.P. Lopez, S.S. Sibbett, ACS Appl. Mater. Interfaces 1 (2008) 124. [19] A.W. Martinez, S.T. Phillips, B.J. Wiley, M. Gupta, G.M. Whitesides, Lab Chip 8 (2008) 2146. [20] K. Abe, K. Suzuki, D. Citterio, Anal. Chem. 80 (2008) 6928. [21] A.W. Martinez, S.T. Phillips, G.M. Whitesides, PNAS 105 (2008) 19606. [22] A.W. Martinez, S.T. Phillips, Z. Nie, C. Cheng, E. Carrilho, B.J. Wiley, G.M. Whitesides, Lab Chip 10 (2010) 2499. [23] J. Wang, R. Pelton, L.J. Veldhuis, C.R. Mackenzie, J.C. Hall, C.D.M. Filipe, Appita J 63 (1) (2010) 32. [24] J. Su, C. Garvey, S. Holt, R. Tabor, B. Winther-Jensen, W. Batchelor, G. Garnier, JCIS 448 (2015) 88. [25] H.P. Fernandes, C.L. Cesar, M.L. Barjas-Castro, Rev. Bras. Hematol. Hemoter. 33 (4) (2011) 297. [26] P. Raj, S. Varanasi, W. Batchelor, G. Garnier, JCIS 447 (2015) 113. [27] P.D. Issett, D.J. Anstee, Applied Blood Group Serology, Montgomery Scientific Publications, Durham, NC, USA, 1998.