blood group typing is necessary in the treatment of patients with massive blood loss . matching the donor 's blood group with the patient 's blood group
the abo blood typing system is tested first for all blood transfusions , because it can cause serious damage in all other blood systems due to its strong specific antigen - antibody interactions .
the abo blood group system is classified based on the inherited properties of red blood cells ( rbcs ) .
it is determined by the presence or absence of certain proteins and oligosaccharides called antigens , including a and b , which are presented on the surface of rbcs .
a , b , and o rbcs structures are similar , but the difference is that a - type rbcs have n - acetylgalactosamine ( galnac ) and b - type rbcs have galactose ( gal ) , while o - type rbcs have neither .
the blood group is associated with anti - a and anti - b immunoglobulin m antibodies ( igm ) , which are the body 's natural defense against foreign antigens
. the abo blood typing system breaks blood types down into four groups : a , b , ab , or o. each group specifies the antigens and antibodies found in that individual , such as the a antigens and b antibodies found in blood group a , the b antigens and a antibodies found in blood group b , both a and b antigens found in blood group ab , and both a and b antibodies found in blood group o [ 15 ] .
surface plasmon resonance ( spr ) technique has become a widely used technique for the detection of biomolecular interactions since the 1990s . among other applications ,
spr sensor is often used in clinical diagnosis for the detection of antigen - antibody binding , protein - ligand interaction , and dna detection [ 612 ] .
spr is a sensitive technique that can be applied for real time monitoring of the biomolecular interactions in solution [ 13 , 14 ] .
the spr biosensor is based on changes of the optical reflectivity by the adsorption of biomolecules on a gold ( au ) surface , which cause a change in refractive index near the spr - active gold surface .
long - range surface plasmon resonance ( lr - spr ) involves surface plasmons ( sps ) that propagate along a thin metallic film embedded between two dielectric materials with similar refractive indices , for which both the evanescent field intensity and the penetration depth ( more than 1  m ) are greater than those for conventional short - range sps ( penetration depth is ~200  nm ) [ 1520 ] .
hence , it is expected that the sensitivity at longer distances from the metal surface will increase with lr - spr spectroscopy .
recently , homola et al . reported that the lr - spr biosensor could detect large analytes , such as the bacteria escherichia coli ( 0.71.0  m ) due to the large penetration depth of the evanescent wave [ 21 , 22 ] .
in addition , the lr - sps enhanced the optical field wave at the metal - dielectric interface , leading to a higher sensor sensitivity and increased penetration into the analyte solution than that observed for conventional spr . as a result , a thicker sensor coating with a significantly larger number of binding analyte molecules on the surface could be used [ 23 , 24 ] .
we have also previously reported an lr - spr immunosensor based on the electrospun poly ( acrylic acid ) ( paa ) fibers for the detection of human igg .
the detection of antigens a and b on the surface of rbcs by conventional spr was reported by quinn et al .
[ 25 , 26 ] . however , the detection of rbcs via conventional spr is limited due to the penetration depth of evanescence field ( ~200  nm ) , which is much less than the size of rbcs ( i.e. , ~2  m thick and ~7.5  m in diameter ) . recently , various studies have been reported in the applications of spr technique for the study of rbcs .
houngkamhang et al .   reported abo blood typing via spr imaging which observed interaction between immobilized anti - a and anti - b antibodies array and a and b antigens on rbcs surface .
the interaction of antigens around lower rbcs membrane surface and immobilized anti - a and anti - b antibodies array was applied to obtain the agglutination strength of the rbcs and immobilized antibody .
the spr imaging , which utilized the shear force generated within the flow cell , was used to measure the rolling speed of red blood cell .
the technique can only extract the information at the interface because the limit of the evanescence field makes it impossible to study in whole cell .
a number of important pieces of information regarding the property of the cell were undetectable such as cell elasticity and cell deformation .
krupin et al . used long - range surface plasmon waveguides for capturing only blood group antigen a on rbcs by immobilized anti - a igg via no comparative studies with anti - b .
to our knowledge , there was no report regarding the detection of rbc by lr - spr . because the difference in surface chemistry , experimental set - up , and nature of spr signal between lr - spr and spr , in this work , it is desirable to explore the possibility of applying lr - spr to detect abo blood group and understand the nature of the lr - spr signal .
anti - a and anti - b will be covalently immobilized on self - assembled monolayer ( sam ) surface and a , b , ab , and o will be detected by using lr - spr .
the cytop ( n = 1.34 ) fluoropolymer is used in the lr - spr system in order to match the refractive index with the phosphate buffered saline ( pbs ) buffer .
all the results of blood typing were consistent with the results obtained from the agglutination test .
the lowest detection amounts of rbc - a and rbc - b were 1.58  10 and 3.83  10  cells / ml indicating that lr - spr has much higher sensitivity than that obtained with conventional spr biosensors [ 25 , 26 ] and lr - spr waveguides . we demonstrate that lr - spr is a good candidate for classification of blood typing and has a high potential in other clinical applications such as bacteria cells , cancer , and rare rbcs .
the 11-mercaptoundecanoic acid ( 11-mua ) , phosphate buffered saline ( pbs ) tablets , and sodium acetate buffer ( ph 5 ) were all purchased from sigma - aldrich .
the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride ( edc ) , n - hydroxysuccinimide ( nhs ) , and ethanolamine hydrochloride ( ea - hcl ) were purchased from tokyo chemical industry ( tci ) .
the ctl-809 m and ctl-180 solvents for the cytop solution were purchased from asahi glass . mixed clones of monoclonal anti -
a and anti - b and standard a cells , b cells , and o cells were obtained from the research unit of the thai red cross society . the kretschmann configuration is used for exciting surface plasmons using he - ne laser with wavelength (  ) of 632.8  nm [ 30 , 31 ] .
lr - spr was arranged to propagate along a thin metallic film embedded between two dielectrics with similar refractive index ( figure 1(a ) ) . for the fabrication of the lr - spr sensor chip , a 7% cytop solution ( dissolved 9% cytop ( ctl-809 m ) in ctl-180 solvent )
was spin - coated on a high - refractive index glass ( ohara s - lah60 , n = 1.83 ) at a first spin rate of 500  rpm for 10  s and second spin rate of 1300  rpm for 20  s. the cytop solvent was dried at 180c for 1  h in an oven , and the cytop film ( ca .
800  nm ) with a refractive index of 1.34 ( similar to a refractive index of water , 1.33 ) was obtained on a high - refractive index glass .
then , 30  nm of gold ( au ) film was deposited on the cytop film by vacuum evaporation . a chromium layer ( 1  nm )
was used to promote au adhesion with the cytop film . as shown in figure 1(b ) , the angular reflectivity curves of the conventional spr and lr - spr chips measured in a bare au / pbs buffer system indicate that the curve of the lr - spr chip is sharper than that of the conventional spr chip .
the lr - spr chip au surface was covered by a self - assembled monolayer ( sam ) using 10  mm of 11-mercapto - undecanoic acid ( 11-mua ) in ethanol .
the carboxylic groups of the sam surface were activated to their ester forms by immersion in 0.4  m edc and 0.1  m nhs dissolved in deionized water at a ratio of 1  :  1 .
the monoclonal antibody for blood group a ( anti - a ) and monoclonal antibody for blood group b ( anti - b ) were covalently immobilized on the activated sensor surface ( after rinsing the sensor chip with pbs buffer ) by injecting the antibodies in sodium acetate at ph 5 at a 1  :  10 dilution onto the activated sam surface .
the residual activated surface sites , which did not react with antibodies , were inactivated or blocked with 0.2  m ethanolamine .
the rbc samples from standard a , b , and o cells were detected on the immobilized antibody by observing the change in the refractive index on the lr - spr biosensor .
standard rbc - a and rbc - b samples were diluted in the range of 3  10 to 3.8  10 cells / ml . the number of cell number was counted using the hematocrit test .
regeneration of the lr - spr chip surface was performed using 20  mm naoh , followed by rinsing with pbs running buffer .
a summary of the antibody immobilization on the biosensor chip and the detection of rbcs is shown in figure 2 .
we compared the sensing ability between conventional spr and lr - spr for the detection and classification of rbc typing on grafted sams ( 11-mua ) .
first , the conventional spr sensor chip was used for the evaluation of the immobilization of anti - a / anti - b on the sensor chip and the detection of rbc - a .
angular reflectivity curves of the conventional spr were observed before immobilization or after grafting the sam , after immobilization of anti - a , and after the detection of rbc - a ( figure 3(a ) ) .
the angular spr reflectivity curve after immobilization of anti - a was shifted to higher angle , indicating that anti - a was immobilized on the sensor chip . the increase in the reflectivity during the immobilization is also shown in the spr kinetic curve ( figure 3(b ) ) .
however , the reflectivity during the detection of rbc - a is almost constant , as shown in the kinetic curve ( figure 3(b ) ) . moreover , the corresponding angular reflectivity curve was almost constant after the detection of rbc - a .
this indicates that the conventional spr sensor is limited to the detection of rbc , presumably because of the fact that the evanescence field of conventional spr is much shorter than the thickness of rbcs , and hence the reflectivity change is not sensitive to the adsorption of the surface .
then , the lr - spr sensor chip was used for the detection and classification of rbc typing on 11-mua sam . the lr - spr kinetic reflectivity curve for the immobilization of anti - a and the detection of rbc - a is shown in figure 4(b ) .
the baseline in the figure is obtained after the activation process by rinsing with pbs buffer .
the reflectivity was obviously increased by the immobilization of anti - a and after the detection of rbc - a ( by ~0.008 ) ( figure 4(b ) ) . in this result
, the baseline of the initial reflectivity is relatively high because the experiment was continuously carried out after the activation of the surface .
hence , the reflectivity was saturated by the injection of anti - a . however , after the detection of rbc - a , we clearly observed the increased reflectivity change compared to the conventional spr . in order to clearly compare with the conventional spr reflectivity curve ,
furthermore , the angular spr reflectivity curve clearly shows that the dip angle shifts to a higher angle after the detection of rbc - a ( figure 4(a ) ) .
these results indicate that the lr - spr is more sensitive for the detection of rbc - a in comparison with the conventional short - range spr measurement .
because one important advantage of the biosensor is reusability , we also studied the regeneration ability of rbc - a on anti - a . for the regeneration of the rbc detection system
, we found that 20  mm of naoh was a suitable condition to disrupt the antigen on the rbcs , causing the disruption of the rbc - antibody specific adsorption interaction without destroying immobilized antibody on the activated surface .
the lr - spr curve is shifted back to the lower incident angle and is almost the same as the lr - spr curve of immobilized anti - a surface , indicating that the adsorbed rbc - a is completely removed from the anti - a surface without removing the immobilized anti - a ( figure 4(a ) ) .  
figure 5 shows the kinetic property of the lr - spr during the regeneration of rbc - a / anti - a surface over three times . during the experiment ,
pbs buffer was used to obtain a baseline reading , which is shown as    in the figure .
after the binding of rbc - a on the anti - a for about 15  min , the surface with residual unbound rbc - a was rinsed with pbs buffer , which is shown as  .  then , 20  mm naoh was injected on the rbc - a / anti - a surface , followed by the injection of pbs buffer ( baseline , shown as    ) . as shown in the figure , each time the baseline returned to the original baseline values , indicating that the anti - a surface still remained ( i.e. , was regenerated ) , even after three sensing and regeneration processes .
next , we immobilized anti - b on activated 11-mua sam surface and detected rbc - b by lr - spr ( figure 6 ) . in this experiment , the sensing procedure was the same as for rbc - a detection described above .
similar to the results for rbc - a , lr - spr showed an obvious increase at each adsorption step , indicating that it can easily detect rbc - b . to study the specificity between antibodies and rbcs ,
anti - a or anti - b was immobilized on activated 11-mua sam surface , and rbc - a , rbc - b , or rbc - o was detected to determine both specific and nonspecific interactions .
the lr - spr kinetic curves during the detection of rbc - a , rbc - b , and rbc - o on anti - a ( figure 7(a ) ) and anti - b ( figure 7(b ) ) show that the reflectivity was increased in all cases .
this indicated that the rbcs were adsorbed on the antibodies both by specific and nonspecific interactions .
after rinsing with pbs buffer , the lr - spr reflectivity for rbc - b and rbc - o decreased to around baseline .
this is because the nonspecifically or physically adsorbed rbcs were removed from the anti - a surface . on the other hand , the lr - spr reflectivity for rbc - a kept higher reflectivity than that of the initial baseline , indicating that the specifically adsorbed rbc - a on anti - a remained on the surface .
the slight decrease by the pbs buffer indicates some physically adsorbed rbc - a was removed from the surface . in the case of the rbcs detection on anti - b surface , the lr - spr reflectivity for rbc - a and rbc - o decreased to almost the initial baseline after the adsorption on the surface , while the reflectivity for rbc - b remained higher .
these results indicate that the specific and nonspecific adsorption of rbcs can be clearly detected by the lr - spr sensor chip , showing the ability to classify blood types .
standard rbc - a and rbc - b samples were serially diluted from their original concentration and injected to study the interaction with anti - a and anti - b immobilized on 11-mua sam .
rbc - a and rbc - b were detected on the immobilized anti - a and anti - b , respectively , by varying each concentration .
rbcs at each concentration were detected for 10  min , followed by rinsing with pbs . the spr reflectivity change ( r )
was obtained by the reflectivity difference between the baseline before the injection of rbcs and after rinsing with the pbs buffer .
antibody surfaces were regenerated with 20  mm naoh for the following experiment . as shown in figure 8 , the reflectivity change ( r ) of lr - spr increases
the limit of detection ( lod ) of rbc - a and rbc - b were at 1.58  10  cells / ml and 3.83  10  cells / ml , respectively .
the lod was defined as three times standard deviation of the blank ( pbs buffer ) .
we found that the obtained lod using the lr - spr is lower than that with conventional short - range spr ( i.e. , 3.3  10  cells / ml ) [ 25 , 26 ] and also lower than that with long - range surface plasmon waveguides that exhibited the lod less than 3  10  cell / ml .
this clearly indicates that lr - spr is a promising technique for the detection of rbcs .
we demonstrated that the lr - spr sensor , consisting of high - refractive index glass , cytop film layer , and thin gold ( au ) film , is capable of detecting large analytes , red blood cell ( rbcs ) .
the antibodies of blood group a and group b ( anti - a and anti - b ) are able to be covalently immobilized on a grafting self - assembled monolayer ( sam)/au surface on the lr - spr biosensor chip . for blood typing , the rbc samples are detected on the immobilized anti - a and anti - b surface by the change in the refractive index .
we found that the results of blood typing obtained by the lr - spr biosensor were consistent with those obtained from the agglutination test .
moreover , the lr - spr exhibited the lowest detection limits of 1.58  10  cells / ml for rbc - a and 3.83  10  cells / ml for rbc - b , indicating that the lr - spr chip has a higher sensitivity than conventional short - range spr biosensors ( 3.3  10  cells / ml ) .
finally , the sensor showed a good efficiency of surface regeneration using 20  mm naoh .
therefore , the lr - spr technique demonstrates many advantages for the detection of rbcs and could be used to perform abo blood group typing in the future .
 in this study , we demonstrate a long - range surface plasmon resonance ( lr - spr ) biosensor for the detection of whole cell by captured antigens a and b on the surface of red blood cells ( rbcs ) as a model . 
 the lr - spr sensor chip consists of high - refractive index glass , a cytop film layer , and a thin gold ( au ) film , which makes the evanescent field intensity and the penetration depth longer than conventional spr . 
 therefore , the lr - spr biosensor has improved capability for detecting large analytes , such as rbcs . the antibodies specific to blood group 
 a and group b ( anti - a and anti - b ) are covalently immobilized on a grafting self - assembled monolayer ( sam)/au surface on the biosensor . for blood typing 
 , rbc samples can be detected by the lr - spr biosensor through a change in the refractive index . 
 we determined that the results of blood typing using the lr - spr biosensor are consistent with the results obtained from the agglutination test . 
 we obtained the lowest detection limits of 1.58  105  cells / ml for rbc - a and 3.83  105  cells / ml for rbc - b , indicating that the lr - spr chip has a higher sensitivity than conventional spr biosensors ( 3.3  108  cells / ml ) . 
 the surface of the biosensor can be efficiently regenerated using 20  mm naoh . in summary , as the lr - spr technique is sensitive and has a simple experimental setup , it can easily be applied for abo blood group typing . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion