Visualization and Quantification of IgG Antibody Adsorbed at the

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Visualization and Quantification of IgG Antibody Adsorbed at the Cellulose-Liquid Interface Vikram Singh Raghuwanshi, Jielong Su, Christopher J. Garvey, Stephen Andrew Holt, Peter James Holden, Warren Jeffrey Batchelor, and Gil Garnier Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00593 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on July 3, 2017

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Visualization and Quantification of IgG Antibody Adsorbed at the Cellulose-Liquid Interface Vikram Singh Raghuwanshi,1 Jielong Su,1 Christopher J. Garvey,2 Stephen A. Holt,2 Peter Holden,2 Warren Batchelor,1 Gil Garnier 1,* 1

Department of Chemical Engineering, Monash University, Clayton, Victoria, 3800,

Australia 2

Australian Nuclear Science and Technology Organization (ANSTO), Locked Bag 2001,

Kirrawee DC NSW 2232, Australia. *Address correspondence to *Email: [email protected]


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ABSTRACT: Quantification of adsorbed biomolecules (enzymes, proteins) at the cellulose interface is a major challenge in developing eco-friendly bio-diagnostics. Here, a novel methodology is developed to visualize and quantify the adsorption of antibody from solution to the cellulose- liquid interface. The concept is to deuterate cellulose by replacing all nonexchangeable hydrogens from the glucose rings with deuterium in order to enhance the scattering contrast between the cellulose film surface and adsorbed antibody molecules. Deuterated cellulose (DC) was obtained from bacterial (Gluconacetobacter xylinus strain) cellulose which was grown in heavy water (D2O) media with a deuterated glycerol as a carbon source. For comparison hydrogenated cellulose (HC) was obtained from cellulose acetate. Both HC and DC thin films were prepared on silicon substrate by spin coating. X-ray reflectivity (XR) shows the formation of homogenous and smooth film. Neutron reflectivity (NR) at the liquid/film interface reveals swelling of the cellulose film by a factor of 2-3 times its initial thickness. An Immunoglobulin G (IgG), used as a model antibody, was adsorbed at the liquid-solid interface of cellulose (HC) and deuterated cellulose (DC) films under equilibrium and surface saturation conditions. NR measurements of the IgG antibody layer adsorbed onto the DC film can clearly be visualized, in sharp contrast in comparison to the HC film. The average thickness of IgG adsorbed layer onto cellulose films is 127 ± 5 Å and a partial monolayer is formed. Visualization and quantification of adsorbed IgG is shown by large difference in scattering length density (SLD) between DC (7.1 x 10-6 Å-2) and IgG (4.1 x 10-6 Å-2) in D2O which enhanced the scattering contrast in NR. Quartz crystal measurements (QCM-D) was used as a complementary method to NR to quantify the adsorbed IgG over cellulose interface.

KEYWORDS: Antibody, IgG, adsorption, Deuterated cellulose, liquid-solid interface, QCM-D, spin coating, X-ray reflectivity, Neutron reflectometry.


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Cellulose is an easy to process bio-degradable polymer containing three labile hydroxyl groups per anhydrosugar monomer, which can be exchanged with other functional groups 1, 2. Lately, cellulose films, paper and even treads have emerged as performant, economic and eco-friendly functional components of diagnostic devices used to identify or quantify specific biomolecules 3, 4, 5, 6. Cellulose based paper diagnostics have been developed for the accurate, rapid and reliable blood typing, glucose measurement and liver function

7, 8, 9

. In these

applications, an immobilized functional protein, such as an enzyme or an antibody (immunoglobulin G or M), releases a signal upon specific binding with the analyte of interest. This signal is proportional to the concentration of the biomolecules immobilized onto the cellulose surface, their availability to bind with the analyte and their ability to react. This means the concentration, distribution, orientation and condition of the immobilized biomolecules onto the cellulose surface are critical variables affecting diagnostic performance. Also important are the adsorption affinity and diffusion of reactants, products and analyte within the cellulose interface. Antibodies and enzymes are by far the most expensive components of paper diagnostics; economics dictate to use as little biomolecules as possible and to maximize their functionality and stability. Efficient engineering of biodiagnostics requires to quantify the adsorbed volume fraction, surface coverage and conformation of the adsorbed biomolecules on the film surface. Intensive efforts presently target quantifying immobilized biomolecules onto cellulose surfaces10. There is a need to simultaneously define biomolecule orientation and surface coverage at the cellulose-liquid interphase. With dimensions ranging from 1 to 15 nm, immunoglobulins (G and M) are very difficult to distinguish from paper made of cellulosic fibers of length 0.8 to 3mm, diameter 8 to 30µm and surface roughness 1 to 2 µm. Further, paper has a tortuous porous void structure of diameter ranging from 1 to 100’s µm. Most modern surface analytical techniques cannot


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detect these biomolecules 3 to 5 orders of magnitude smaller than the critical heterogeneity of the paper substrate. A more efficient approach which removes the effect of physical heterogeneity consists of replacing paper by quasi molecularly smooth cellulose films. Multiple experimental methods can characterize thin film morphology and the adsorption of 11, 12

macromolecules on the film. These include X-ray

(XR) and neutron reflectivity (NR),

atomic force microscopy (AFM), surface plasmon resonance (SPR) and Quartz Crystal Microbalance (QCM). Each method has intrinsic limitations such as radiation damage, resolution and contrast between substrate and adsorbed macromolecule. NR is a nondestructive and powerful technique to characterize thin films multilayers and biomolecules adsorption at the solid-liquid/gas interface

13, 14

. Hydrogen is one of the main atomic

component of biomaterials and there is a large difference between the neutron scattering cross sections of H and D. Neutrons are sensitive to variations in the scattering length density (SLD), or the sum of atomic cross sections of atoms in the material15. Thus, by replacing H with D (or vice versa) in materials and probing them with neutrons, it is possible to vary the scattering contrast which enables the visualization of thin layers of molecules adsorbed at the substrate- liquid/solid interface. One of the problems in characterizing the morphology of biomolecules adsorbed onto cellulose surfaces using neutrons is the small SLD difference (contrast) between the two phases (cellulose and biomolecules). Two approaches can be employed to enhance the SLD difference: deuterated biomolecules or cellulose surface. In deuterating biomolecules, it is difficult to determine the deuteration level due to the unknown information on the labile H in biomolecules. Deuterating cellulose provides a better option as chemical structure and labile H’s are well known; further, the adsorption conformation of any biomolecules can then be quantified.


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Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) is a complementary method to reflectivity and an analytical method to monitor in-situ adsorption of molecules14. In QCM-D, changes in the resonance frequency of quartz crystal sensor (5 MHz) are monitored with respect to the amount of molecules adsorbed over the crystal surface16. It quantifies the adsorbed mass in the limits of nanograms and reveals the respective thickness and surface coverage. The dissipation mode of the QCM-D measures the viscosity and the rigidness of the layer adsorbed over the sensor surface. The objective of this study is to quantify the morphology and the amount of antibody molecules physisorbed from aqueous solution onto the cellulose-liquid interface. Smooth and thin deuterated cellulose (DC) films were prepared to enhance the contrast between cellulose surface and adsorbed antibodies, thus providing biomolecule visualization. Both hydrogenated (HC) and deuterated (DC) cellulose films were made by spin coating cellulose derivatives followed by regeneration into the pure cellulose form. XR measurements analyse the quality of film with respect to film thickness and roughness in air. An IgG antibody immunoglobulin G (IgG) was selected as model protein to adsorb onto the cellulose film surface

17, 18

; it is one of the common antibodies used in human blood typing. NR

measurements reveal the contrast obtained from the adsorption of IgG onto the protonated (HC) and deuterated (DC) cellulose films. The large difference in SLD between IgG and DC allows one to visualize the IgG adsorbed onto the surface of DC films. Modelling NR data permits the quantification of IgG in terms of layer thickness, volume fraction and surface coverage. To complement, Quartz Crystal Microbalance with dissipation (QCM-D) quantified the amount, reversibility and kinetics of IgG adsorption at the cellulose-liquid surface. Here, we aim at developing robust, low cost and accurate cellulosic biodiagnostics by better understanding the behaviour of the functional biomolecule at the cellulose-liquid interface.


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EXPERIMENTS Materials Cellulose acetate (CA), 1-Butyl-3-methylimidazolium chloride (BMIM chloride, C98.0%, HPLC), Hexamethyldisilazane (HMDS, ReagentPlus, 99.9%), toluene (anhydrous, 99.8%), ethanol and acetone (AR, C99.5%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polished silicon blocks [50.8 mm diameter and 12 mm height, n-type Si:P (100)] were purchased from SIL’TRONIX (Archamps Technopole, Archamps, France). Silicon blocks were cleaned in ammonium hydroxide, hydrogen peroxide and water (volume ratio = 1:1:5) at 70 °C for 15 min before used for film deposition. Spin coating was performed with a WS650-23B spin coater (Laurell Technologies Co. North Wales, PA, USA).

Cellulose film preparation Hydrogenated cellulose (HC) films were obtained by first spin coating cellulose acetate (CA) dissolved in acetone on polished silicon blocks. Later, CA films were regenerated into cellulose by keeping the films in sodium methoxide solutions as reported earlier by Su et al 19. Deuterated cellulose (DC) films were obtained by using deuterated bacterial cellulose (DBC) produced from Gluconacetobacter xylinus strain ATCC 53524 grown in D2O media with a deuterated glycerol carbon source20. DBC was first dissolved in ionic liquid (BMIM chloride) for 6 h. Then HMDS was added in the solution and the mixture stirred at 100 °C for 2 h, this is followed by the addition of the anhydrous toluene and washing with the anhydrous methanol. Drying gives a white amorphous solid powder of deuterated trimethylsilyl cellulose (TMSC). Finally, the TMSC powder is dissolved in toluene and the solution is spin coated onto the Si blocks. The TMSC films were hydrolysed back into DC films by exposing to HCl vapours (1 M) in a closed container for 15 mins. Further details of the DBC film preparation is published elsewhere 10.


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Reflectivity measurements XR measurements were performed at the solid/air interface on a Panalytical X'Pert Pro instrument with Cu Kα X-ray source (λ = 1.54 Å). NR measurements were performed at the PLATYPUS time-of-flight reflectometer at the 20 MW OPAL reactor of the Australian Nuclear Science and Technology Organization (ANSTO, Sydney, Australia) nuclear facility. The wavelengths of cold neutrons ranging from 2 to 20 Å. Measurements were performed at three different angles (0.5, 0.85 and 3.8 degrees). The sample to detector distance was 2500 mm. The area detector 3He gas, 2D 'Denex' (500mm wide by 250 mm high. 0.7 mm vertical resolution, 2.5 mm horizontal resolution) was used to collect reflected neutrons. The wavelength resolution and slit collimation applied to the incident neutron beam results in a ΔQ/Q value of 3.2 %. Data reduction and modelling of the NR curves were made by using the Igor based software MOTOFIT

21

. During fitting a pre-formed layer of SiO2 on the bare Si block was included.

NR curves were fitted with single and two layer models. In both cases the thickness, scattering length density (SLD), roughness and solvent fraction were the fitting variables for the cellulose film layer and the adsorbed biomolecule layer on film. Moreover, a constant background was included. QCM measurements QCM measurements were performed using an E1-QCM-D instrument from Biolinscientific Ltd. Quartz crystal sensors coated with cellulose was used as supplied. The cellulose sensor was mounted into the flow cell and changes in the frequency (F) and dissipation (D) with respect to the fundamental frequency of 5 MHz and six different odd overtones (1, 3, 5, 7, 9 and 13) were simultaneously monitored. First, a stable base line in film/air interface was obtained than the buffer solution (0.9% NaCl-H2O) was passed through the flow cell. The


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cellulose film swells into the buffer solution. Later, IgG dispersed in buffer solution (concentration 1 mg/mL) was passed through the sensor. Once stability in the change of F was observed, the sensor was again flushed with the pure buffer solution. Obtained curves were fitted by applying a viscoelastic model using the software QSense Dfind from Biolinscientific Ltd.

RESULTS Characterization of cellulose thin films. Regenerated HC (from CA) and DC thin films (from DBC) were characterized by X-ray reflectivity (XR). The XR curves of the HC and DC films are shown in Figure 1a and 1b, respectively. Both films were measured at the cellulose/air interface. Multiple fringes in both reflectivity curves reveal smooth and uniform films of low roughness. The thickness and roughness of these films were determined by fitting the XR curves using a single cellulose layer model with the Igor based macro MOTOFIT

21

. The solid lines in Figure 1 show the model fit of reflectivity profile as a

function of the q vector (q=

) where, λ is wavelength of the incident neutron beam and

θ is the incident angle, and the respective scattering length density (SLD) profile with respect to film thickness is given in the inset.


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Figure 1: a) XR curve of the regenerated deuterated cellulose film obtained from deuterated bacterial cellulose. b) XR curve of the regenerated hydrogenated cellulose film obtained from cellulose acetate. The solid line show the fit using a single layer model and the inset represents the respective SLD profile obtained from XR curve fitting.

Table I shows the film parameters obtained from fitting the XRR curves. The thickness of the SiO2 layer is about 18 ± 4 Å. The evaluated thickness of HC film is 205 ± 5 Å, which is about twice that of the DC film (80 ± 4 Å). The surfaces of both cellulose films are smooth and suitable for measuring IgG adsorption. Table I: Cellulose film thickness and roughness resulting from XR curves fitting of HC (from CA) and DC (from DBC). Sample

Thickness (Å)

Roughness (Å)

HC film

205 ± 5

12±3

DC film

80 ± 4

15± 3


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Adsorption of IgG onto HC and DC films. The adsorption of IgG molecules from solution onto HC and DC thin film surface was performed by first dispersing the IgG molecules at 1mg/mL into a 0.9%NaCl-D2O buffer solution subsequently flowed in large excess over the film surface. These conditions insure the excess availability of IgG for surface saturation. NR measurements serve to quantify the adsorption of IgG molecules onto the HC and DC films. Figure 2 shows the NR curves for the HC film (black) and IgG (green) adsorbed over HC film measured at the solid/liquid interface. No significant difference is observed between the NR curves of HC film with and without the layer of adsorbed IgG. The NR curves were fitted with the single layer model for the cellulose film. The obtained parameters show the thickness of the cellulose film to be about 386 Å in the buffer solution, almost twice the initial film thickness (205 Å). Water penetrates within the chains of the cellulose layer to relax the non-equilibrium structure conformation formed during the film spin coating process; this results in film swelling

14, 16

. The SLD values obtained from fitting the HC film curve

(C6H7D3O5 in D2O buffer) is 3.7 x 10-6 Å-2 assuming three liable hydrogens and the SLD for IgG to be 4.1 x 10-6 Å-2. This minute difference of SLD observed between HC and IgG reflects the poor contrast shown in the RQ4 vs Q plot in the supporting information S1. The observed contrast is not sufficient to accurately quantify the IgG layer adsorbed onto the HC film surface. Therefore, there is a need to further differentiate between the SLDs for quantification.


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Figure 2: Experimental NR curves of the hydrogenated cellulose (HC) film (Black) and the HC/IgG (Green) measured in D2O-0.9%NaCl buffer. DC films were prepared to enhance the contrast (SLD difference) between cellulose and IgG molecules to visualize and quantify the adsorbed IgG molecular layer using NR. The DC film properties were also measured at the air/film interface with neutrons and fitted with the single layer model (supporting information S2). The parameters evaluated by curve fitting with the model were film thickness, roughness and SLD (Table II). The evaluated DC film thickness is 80 Å with a roughness of 15 Å. The thickness obtained from NR and XR analysis are in good agreement. The resulting SLD value of the DC film (in air) is 5.6 x 10-6 Å-2 which corresponds to cellulose C6H3D7O5; this represents the fully deuterated cellulose (C6D10O5) having the three labile Ds from hydroxyl exchanged with Hs from the atmosphere. The theoretically calculated SLD value for the composition of cellulose C6H3D7O5 is 5.56 x 10-6 Å-2.


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Further, NR measurements were made on DC film at the film/liquid interface at two different contrasts (0.9%NaCl -100%D2O and 0.9%NaCl- 70%D2O30%H2O). The cellulose film swells due to the penetration of the aqueous buffer within the structure of the film. The green NR curve in Figure 3a and the blue NR curve in Figure 3b show the DC film at two different contrasts. Multiple fringes in the NR curves reveal good film uniformity. The curves were fitted with a single layer model with the parameters presented in Table II. The thickness of the DC film (in buffer) is 198 Å, about 2.4 times the thickness (80 Å) measured at the air/film interface due to swelling. Moreover, the SLD of the DC film is 7.2 x 10-6 Å-2 in D2O buffer and 6.7 x 10-6 Å-2 in D2O-H2O buffer corresponding to SLD for chemical composition of C6D10O5 and C6H1D9O5, respectively. The film volume fraction of cellulose obtained from NR curve fitting is 90% and for solvent is 10 %. This volume fraction of cellulose does not correspond well to the increase in film thickness measured due to solvent penetration. Volume fraction of the solvent in the film can also be evaluated with respect to the change in film thickness by 22:

=

(1)

Where, Ti is the initial thickness and Tf is the final thickness. Here, Ti =80 Å; Tf =198 Å for which the calculated volume fraction of solvent is ρsol =0.6 (60%) and the volume fraction of cellulose ρcel=(1- ρsol) is 0.4 (40%). The volume fraction evaluated by the latter method is more convincing that the former fitting procedure. IgG molecules were dispersed in the buffer (at 1 mg/mL) and adsorbed by flowing the solution onto the DC film surface under excess conditions. NR curves for the DC film with the adsorbed IgG layer were also measured in two different contrast buffers: 0.9%NaCl/D2O (Figure 3a; red curve) and 0.9%NaCl/70%D2O-30%H2O (Figure 3b; brown curve). In both Figures, multiple fringes were observed for the pure DC film which is directly connected with the film thickness, smoothness and difference in SLD. Direct adsorption of IgG onto the


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DC surface can be clearly seen by the shift of the NR curve towards lower q values which reveals an increase in film thickness due to the adsorption of the IgG antibodies molecules.

Figure 3: (a) NR curves of the deuterated cellulose (DC) film measured in D2O (green) and the adsorbed IgG antibody layer (red) on the film. The NR measurements were performed in NaCl-D2O buffer. (b) Same NR measurement of the DC and DC/IgG in NaCl-70% D2O –30% H2O buffer. The blue sphere curve in (b) is from pure deuterated cellulose and the brown star is from the adsorbed IgG. Valuable information can be extracted from the NR curves of DC/IgG. An additional layer was fitted for IgG to the DC layer as a fitting variable. Figure 4 shows the fitted NR curves for the DC and DC/IgG films (solid lines) and Table II summarizes the resulting parameters. The thickness of the adsorbed layer of IgG measured in D2O buffer is 129 Å and 126 Å in 70%D2O-30%H2O. Figure 4(b) compares the SLD variation with respect to thickness from NR curve fitting for the two different contrasts. In D2O, the resultant SLD difference between IgG and DC surface is large compared to that from the 70%D2O-30%H2O buffer. The


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evaluated SLD for the adsorbed IgG layer are 4.1 x 10-6 Å-2 and 3.8 x 10-6 Å-2 in D2O and 70%D2O-30%H2O, respectively (Table II).

Figure 4: (a) Deuterated bacterial cellulose with and without antibody (IgG). Both curves are measured in D2O buffer. Solid line shows the NR fit. (b) SLD evaluated from fitting the NR curves.

Table II: NR curve fitting parameters for the DC film measured in air, D2O buffer and with the adsorbed IgG layer. Sample

Thickness (Å)

SLD -6

-2

(10 Å )

Volume

Cellulose

Roughness

Fraction of

composition

(Å)

cellulose (%) DBC – Air

80 ± 4

5.6

78

C6H3D7O5

15± 3

DBC - D2O

198 ± 5

7.2

90

C6H0D10O5

8± 3

DBC-70%D2O-

197± 5

6.7

90

C6H1D9O5

8± 3

129± 5

4.1

15 (IgG)

---

12± 3

30%H2O IgG D2O


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IgG- 70%D2O-

126± 5

3.8

15 (IgG)

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---

18± 3

30%H2O

Adsorption of IgG on cellulose film surface by QCM-D. Quartz crystal microbalance with dissipation (QCM-D) measurements were performed to quantify the adsorption of IgG using a HC cellulose coated sensor surface. Absorption of water in the film and IgG onto the surface were monitored with the variation in quartz crystal frequency which dissipates with respect to the mass adsorbed onto the crystal surface. QCM measurement was performed on the cellulose sensor by measuring first at the 0.9%NaCl/cellulose interface until equilibrium was reached (flat baseline). As the buffer solution absorbs into the cellulose film, swelling occurs. Change in frequency (ΔF) and dissipation (ΔD) with respect to the fundamental frequency of 5 MHz and six different odd overtones were simultaneously monitored. IgG (1 mg/mL) dissolved in 0.9%NaCl-H2O buffer is then passed through the flow cell which allows IgG molecules to adsorb onto the cellulose surface until saturation. Injection of IgG decreases the ΔF due to the adsorption of IgG onto the cellulose surface. A stable plateau was reached when the IgG molecules saturated the film; the sensor surface was then rinsed with the buffer solution to remove all non-adsorbed IgG molecules and shift the equilibrium until a new stable plateau was reached. Figure 5 shows the evolution in F1:3 and D1:3(inset) as a function of time for the adsorption of IgG (IgG/0.9%NaCl) onto the cellulose surface followed by a rinse cycle with 0.9%NaCl. The average change in frequency ΔF1:3 due to IgG adsorption is about ΔF1:3 =75 (after rinse with NaCl).


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Figure 5: a) Frequency variation of F1:3 overtone from QCM-D measurement. Inset shows the change in the dissipation D1:3. The thickness of the IgG layer adsorbed onto cellulose was evaluated by fitting the QCM data of adsorbed of IgG. A visco-elastic model was selected to fit the variation in overtone and their respective variation in dissipation as given in the supporting information S3. The evaluated IgG layer thickness on cellulose film is 140 ± 5 Å and the adsorption kinetics is shown in Figure 6.


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Figure 6: Evolution of adsorbed IgG layer thickness on the cellulose sensor as a function of time. Results obtained from fitting the QCM data from Figure 5a.

DISCUSSION Neutron reflectivity measurements are sensitive to the neutron scattering length density (SLD) distribution while X-rays are scattered based on the electron density distribution 23, 24, 25. The uniquely large scattering cross section difference between H (-0.37 x 10-4 Å) and D (0.67 x 10-4 Å) allows varying contrast in the system by replacing H with D or vice versa. Exchanging H and D has no effect on the chemical structure and only alters the SLDs. A certain level of deuteration is achievable simply by equilibrating the sample into D2O to allow the labile H to exchange. Cellulose has a chemical composition of C6H10O5 with three labile –OH groups per monomer; this confers a SLD of 1.7 x 10-6 Å-2 for cellulose. Exchanging the three labile H with D (in D2O) alters the cellulose composition to C6H7D3O5 which enhances the SLD to 3.4 x 10-6 Å-2. Furthermore, the SLD of proteins in D2O ranges between 2 x 10-6 Å-2 and 4 x 10-6 Å-2 depending on the liable H in the protein structure 26.


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The minor difference in SLD between the cellulose and adsorbed IgG produces insufficient contrast to visualize and quantify IgG adsorbed at the cellulose interface with neutrons (Figure 2). Deuterating either IgG molecules or cellulose can enhance the SLD difference (contrast). However, deuterating IgG is difficult to achieve, is biomolecule specific and does not provide reliable information on the labile H atoms so the respective SLD calculation remains challenging. Conversely, cellulose with its ten H atoms per monomer provides attractive possibilities in monitoring and of easy exchange of H for D to enhance and vary the SLD contrast. Deuterating cellulose enables the enhanced visualization of any adsorbed protein- such as antibodies (IgG, IgM) and enzymes, representing the bulk of functional biomolecules in paper diagnostics and low cost testing devices. Deuterated cellulose (DC) films were made from deuterated bacterial cellulose (DBC). Previously reported ATR-FTIR measurements revealed the complete exchange of H with D in DBC10. The SLD of fully deuterated cellulose (C6D10O5) film is 7.2 x 10-6 Å-2 and exhibits three labile D per monomer. These liable Ds exchange easily with the naturally available H of the atmosphere. The SLD obtained for DC film after fitting the NR curve (in air; Table II) is 5.6 x 10-6 Å-2 which corresponds to the theoretically calculated SLD value (in air) of 5.58 x 10-6 Å-2 for DC (C6D7H3O5). The evaluated SLD value for IgG is 4.1 x 10-6 Å-2 (in D2O) and 3.8 x 10-6 Å-2 (in 70D2O30H2O). The large difference between the SLD of IgG (4.1) and DC (7.2) allows differentiating, visualizing and quantifying IgG molecules adsorbed onto the cellulose surface. The average thickness of the adsorbed IgG layer determined from fitting the NR curve is 127 Å for a volume fraction of 15 %. In Figure 4b, the slope of the SLD profile at the cellulose/IgG interface is not sharp, potentially indicating a transition zone between two layers. This suggests that IgG molecules may penetrate into the swollen cellulose film.


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The IgG antibody molecule is a “Y” shaped molecule of molecular weight of 150 kDa and size: 130 Å x 80 Å x 20 Å. Surface coverage area for the IgG molecules can be calculated by the equation given as 19, 27:

Γ=

(2)

Where Mw is the molecular weight, ɸ is the volume fraction, T is the thickness, Na is the Avogadro number and V in the molecular volume of an IgG molecule. A full surface coverage area (ɸ=1, T= 127 Å) for IgG molecules is expected at about 15 mg/m2. NR results reveals the 127 Å thick IgG layer to be made of only 15 % IgG molecules and 85 % water. The average surface coverage value for IgG molecules on DC film surface is 2.3 mg/m2 (using Eq. 2; ɸ=0.15, T= 127 Å) and the respective average number of IgG molecules per unit area is approximately 1016/m2. Based on a completely different mode of detection, the complementary QCM-D experiments show the formation of a 140 Å thick layer of IgG adsorbed onto the cellulose coated quartz sensor. The respective IgG mass adsorbed onto the cellulose surface was calculated by monitoring the change in the ΔF =75 and by using the equation

∆ =

.∆"

(3)

#

where n=3 is the 3rd overtone, C=0.177 mg/m2

16

. The calculated surface coverage of IgG

adsorbed on cellulose film is 4.2 mg/m2 which correspond to 25 % of the volume fraction of adsorbed IgG. A difference in the surface coverage is observed for the adsorbed IgG on the DC film (2.3 mg/m2) and the cellulose coated sensor (4.2 mg/m2). This is very likely because of the different sources of cellulose material used for the thin film preparation. In this investigation DBC was used, obtained from Gluconacetobacter xylinus strain ATCC 53524 grown in D2O media

28

; also the bacterial cellulose was reacted into TMSC, dissolved in THF and spined

coat and regenerated by HCl hydrolysis. The commercial cellulose coated on quartz sensor is


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from softwood cellulose pulp which is microfibrillated cellulose (Fibril diameter 5-6 nm) and fibril aggregates of 10-20 nm 29. Different source of cellulose exhibits different crystallinity, surface area and cellulose phases which result in the formation of different thin film morphologies. The crystalline/amorphous phase ratio in cellulose thin film strongly affects the swelling behaviour of film and very likely the respective adsorption phenomenon of proteins. This is a subject still poorly understood which deserves further exploration.

CONCLUSION Thin and smooth deuterated cellulose (DC) films were prepared from deuterated bacterial cellulose. The adsorption of IgG at the solid-liquid interface was studied by neutron reflectivity on DC and hydrogenated cellulose (HC) films. XR measurements show that both types of cellulose films are smooth and of low roughness. The dry HC and DC films are 205 Å and 84 Å thick, respectively. These films swell in water. NR measurements at the cellulose/liquid interface show the DC film swells to 198 Å, 2.4 times its dry film thickness. The HC film swells to 386 Å, about 1.9 thicker than its dry thickness. Comparing the NR measurements of IgG adsorption on HC and DC reveals that the DC film provides an excellent contrast, sufficient to visualize the adsorbed IgG antibody molecules. The SLD obtained for the HC (C6H7D3O5) with three labile H in D2O is 3.67 x 10-6 Å-2, while the SLD of IgG is 4.1 x 10-6 Å-2 which shows only a small SLD difference (contrast). However, the SLD of the DC (C6D10O5) film is 7.2 x 10-6 Å-2 in D2O, which demonstrates a sufficiently large SLD difference (contrast) with respect to the IgG layer for accurate measurements. The average layer thickness of the adsorbed IgG antibodies is 127 Å with a volume fraction of 15 % and a surface coverage of 2.3 mg/m2. QCM measurements, which is complementary to NR, also reveals the adsorption of IgG layer to be140 Å thick and the surface coverage to be 4.2


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mg/m2 with 25 % of volume fraction onto the cellulose film. Using deuterated cellulose allows visualization and quantification of any adsorbed protein based biomolecules, such as antibodies and enzymes, onto cellulose surfaces. This knowledge helps understanding the interaction between cellulose surface and functional biomolecules to improve the performance of paper diagnostics, the longevity of antibodies and the efficiency of physioabsorbed biomolecules used in low-cost cellulosic biosensors.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Neutron reflectometry measurements on the hydrogenated and deuterated cellulose film with and without adsorbed IgG layer. QCM-D overtone fitted with a viscoelastic model.

ACKNOWLEDGEMENTS Funding was provided by ARC LP09990526. Thanks to ANSTO and AINSE for beam time (Proposal Number 4654) on the neutron reflectometer PLATYPUS and providing deuterated cellulose; and travel grants, respectively. The work conducted at the National Deuteration Facility (http://www.ansto.gov.au/ndf) is partially funded by the National Collaborative Research Infrastructure Strategy, from the Australian Government. Thank to Dr. Natasha Yeow for IgG preparation.

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Visualization and Quantification of IgG Antibody Adsorbed at the

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