13 Sensitive antifouling surfaces
13.4 Sensing platforms in real biological media
CHAPTER 13.SENSITIVE ANTIFOULING SURFACES
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The novel copolymer brushes, poly(MeOEGMA-b-CBAA-3), allowed facile and fast functionalisation with neutravidin. Immobilisation of streptavidin was considerably lower than that of neutravidin (Figure C13-7 A). Importantly, contrary to pure poly(CBAA-3) brushes, the immobilised neutravidin on the block copolymer showed high activity which was exploited for immobilisation of biotinylated RαM IgG antibody or a biotinylated oligonucleotide (ONC-1). The activity of these receptors was confirmed by detection of mouse IgG (M IgG) (Figure C13-7 B) or by the hybridisation with the complementary oligonucleotide conjugated with BSA (BSA-ONC-2) (Figure C13-7 C). Remarkably, after all the immobilisation steps and detection no fouling from blood plasma was observed (Figure C13-7 B).
The copolymer system represents a novel alternative to pure poly(CBAA-3) brushes.
The well controlled ATRP of the first block allows to easily tune the thickness while the very thin poly(CBAA-3) block accounts for the excellent resistance to fouling and ease of functionalisation without impairing the biological activity of the bioreceptors.
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formulas (Appendix III). Poly(HEMA) brushes, showing complete resistance to fouling from milk samples, were activated with DSC (0.1 M) and N,N-dimethylamino pyridine (0.1 M) and functionalised with anti(CB).
Concentrations of Cronobacter down to 106 cells·mL-1 could be detected in spiked samples of whole fat milk Madeta, powder milk Laktino preparation, and PIF Sunar preparation without any correction for non-specific response to fouling (Figure C13-8).
The specificity of the sensor was assessed by measuring the milk samples spiked with Yersinia enterocolitica 108 cells·mL-1. No cross-interaction was observed.
Figure C13-8. The detection of Cronobacter at concentration of 108, 107 and 106 cells·mL
-1 in 3 types of milk samples by biosensors based on anti(CB) attached onto poly(HEMA) brush. The bacteria were spiked in (A) whole fat milk, (B) Sunar – PIF preparation, and (C) Laktino – powder milk preparation.
The prepared sensor represents the first example of the use of polymer brushes for sensing bacteria and of direct in real time detection of Cronobacter. Remarkably, the approach is generic, thus it can be applied to the preparation of biosensors for detection of various food-borne pathogens.
13-12 13.4.2 D
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In immunocompetent individuals only three diagnoses are relevant: i) primary infection as cause of mononucleosis, ii) a past infection that excludes mononucleosis and iii) the absence of EBV which indicates susceptibility. These possible diagnoses can be correlated with the presence of specific serological markers (Figure 9 and Table C13-2).[37] A primary infection is characterised by the presence of immunoglobulin M and G against viral capsid antigen (VCA) complex, immunoglobulin G against early antigen (EA-IgG) and absence of IgG against Epstein-Barr nuclear antigen (EBNA-(EA-IgG). However, the presence of VCA-IgG and EBNA IgG and the lack of EA-IgG confirms a past infection.
Negative results for VCA-IgG, VCA-IgM, EA-IgG and EBNA-IgG indicate that the individual has not been infected.
Table C13-2. Interpretation of EBV-specific serological profiles for diagnoses.[37]
EA-IgG VCA-IgM VCA-IgG EBNA-IgG
No infection - - -
-Primary infection + + +
-Past infection - - + +
Currently, typical tests for EBV include: heterophile test, indirect fluorescent antibody assay (IFA), Western blot, enzyme linked immunosorbent assay (ELISA) and PCR analysis. All these methods require highly trained personnel and take from few days to several hours to yield results, while not supporting any automation. Furthermore, heterophile test and ELISA suffer from cross-interactions resulting in false positives (ELISA) and false negatives (heterophile test). IFA still remains the gold standard, however, it requires up to 8 days which can compromise the life of the some patients.[37]
Therefore, new methods ensuring fast and direct detection while supporting automation are highly necessary. SPR platforms emerge as the most promising biosensor technology allowing fast and label-free direct detection of biomarkers. However, detection in clinical samples of serum requires optimised bioactive antifouling surfaces.
13.4.2.1 Preparation of the biosensor and optimisation of sensing conditions
The diagnosis of EBV infection is carried out by the detection of specific markers (VCA antibodies, EA-IgG and EBNA-IgG) present in human serum. The strategy to achieve this was to combine the fouling-resistant poly(HOEGMA) brushes with immobilisation techniques allowing to anchor specific bioreceptors (antigens: VCA, EA and EBNA) with spatial resolution to prepare SPR biochips.
The preparation of the biosensing SPR surface is shown schematically in Figure C13-10. 30-nm-thick poly(HOEGMA) brushes were grown from a gold-coated SPR surface by surface initiated ATRP. Streptavidin was covalently immobilised via activated hydroxyls in the brush as described in 13.2.2.[10] Subsequently, four biotinylated
CHAPTER 13.SENSITIVE ANTIFOULING SURFACES
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oligonucleotides (Table C13-3) were linked to the surface of four independent channels of the biochip via biotin-streptavidin interactions. The complementary oligonucleotides with bovine serum albumin conjugated with up to 15 antigen molecules (ONC-1, 2 and 3) and a reference unconjugated oligonucleotide (ONC-4) were hybridised to prepare a biochip with 3 sensing and one reference channel.
The tested serum was driven simultaneously through the 4 channels and the binding of the biomarkers is monitored in real time. After detection, the biosensor can be regenerated by heating above the melting temperature of the ONCs (43ºC) which induces cleavage of the hydrogen bonds between complementary ONC strands. The regenerated oligonucleotides can be subsequently used for the hybridisation of new bioreceptors.
Figure C13-10. Scheme showing the preparation of the biosensors for detection of EBV infection. (A) Immobilisation of streptavidin on poly(HOEGMA) brushes, (B) immobilisation of biotinylated oligonucleotide, (C) hybridisation of complementary oligonucleotide with specific antigen (red circle), (D) detection of EBV markers (antibodies) in clinical serum and (E) regeneration of the sensor by heating to 50 ºC.
Table C13-3. Oligonucleotides used to prepare the biosensor Biotinylated
ONC
Sequence Complementary
ONC
Antigen conjugated
ONC-1 5´-CTACTGAAATACACAGAA-3´ ONC-1’ VCA
ONC-2 5’-GTGTAGATCTTAACGAGT-3´ ONC-2’ EA
ONC-3 5´-GGTACTATACTATGGTCTC-3´ ONC-3’ EBNA
ONC-4 5‘-TTCTGTGTATTTCAGTAG-3´ ONC-4’
-Regeneration 40-50ºC
(A) (B) (C) (D) (E) (F)
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Figure C13-11. (A) Preparation of the biosensing platform. Biotinylated ONC-1, 2, 3, and 4 (curves 1, 2, 3 and 4) are immobilised from their 0.5 μM solutions in TRIS on poly(HOEGMA) functionalised with streptavidin. Subsequently, hybridisation with their complementary ONC-1’,2’ and 3’ was carried out (curves 1, 2 and 3). On curve 4, ONC-1’
is contacted with ONC-4 immobilised on pol(HOEGMA)-streptavidin. No cross-interaction between non-complementary oligonucleotides is observed. (B) Sensor response to positive (1) and negative (2) sera (1 % in PBS).
Figure C13-11 (A) shows the immobilisation procedure for 3 sensing channels, where ONC- 1, 2 and 3 and their complementary ONCs were immobilised from 0.5 μM solutions in TRIS buffer (pH 7.2). When a non-complementary ONC was contacted with the surface (curve 4, ONC-4 immobilised on poly(HOEGMA) contacted with ONC-1’), no cross-interactions were observed. Figure C13-11 (B) shows a typical sensor response to 1
% sera, positive (1) and negative (2). At 1 % or lower dilutions of blood serum, negligible fouling was observed.
The concentration of clinical serum samples was optimised to avoid false negatives and positives and minimise sample consumption. A high concentration of serum yields higher sensor response, however, it may lead to some fouling on poly(HOEGMA) brushes.
On the other hand, excessive sample dilution hampers the detection of the markers. In order to select an optimal concentration, the sensor response for each antigen was measured as a function of the concentration of positive serum. Figure C13-12 (A) shows typical SPR traces for the detection of EBNA-IgG on concentrations of serum ranging from 0.05 to 10 % in TRIS buffer. Concentrations of serum as low as 1 % were sufficient to achieve a clear sensor response (Figure C13-12 (B)) while no fouling was observed (Figure C13-11 (B)). Thus, this concentration was used for all the subsequent analyses of clinical samples.
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Figure C13-12. Sensor response: (A) SPR traces for various concentrations of serum positive to EBNA diluted in TRIS. (B) Sensor response for various concentrations of sera positive to EBNA (black curve), VCA (red curve) and EA (blue curve) markers. (Response of the reference channel was subtracted).
The immobilisation procedure described above is being used for the preparation multispot biosensor arrays. The sensors are currently being tested in the Institute of Photonics and Electronic, Academy of Science of the Czech Republic, v.v.i.