despite great advancements in genomics research , dna microarrays are still of limited 
 applicability as diagnostic tests , in part because proteins , not genes , are the ultimate effectors of a biological process .
proteins are often expressed at concentrations and varied 
 structural forms having modifications that can not be predicted from mrna analysis .
there is a great deal of interest in proteomics - based approaches that enable profiling the abundance of proteins , or investigation of protein - protein and protein - drug interactions .
the concept of a protein microarray or biochip is attractive for rapid , high - throughput proteomics studies that utilize available sample volumes [ 1 , 2 , 3 , 4 ] .
proteins that are present in abnormal concentrations ( increased or decreased ) in a disease state could be identified and validated as disease markers .
proteomics pattern analysis based on a multiplexed list of biomarkers may well make possible the diagnosis of certain diseases that do not have either effective screening options or biomarkers already detected by conventional immunoassays .
identification of such disease markers will provide 
 valuable information for detection , classification , and prognosis of diseases , as well as targets for treatment outcomes .
high - throughput protein chips require arraying a wide range of probes that specifically recognize a single protein in complex mixtures such as serum , plasma , and other biological specimens .
these probes are most frequently antibodies or antibody mimics [ 3 , 4 , 6 , 7 ] . given the immense promise of proteomics technologies , there are still limitations that need to be overcome , such as lack of sensitivity and enhanced information about the target .
because there is no pcr equivalent available to amplify proteins , the identification of low - abundance proteins may often be difficult or impossible , and important biomarker information could thus be lost . rolling - circle amplification ( rca ) is a unique signal amplification technology   that permits detection of multiple proteins with a broad dynamic range on protein microarrays [ 4 , 9 ] . in this paper
, we describe the development of rca - based highly multiplexed and sensitive protein microarray immunoassays for detecting 150 proteins in an elisa format .
application to human serum and plasma samples has established that the rolling - circle amplified protein microarray is a powerful tool for the profiling of expressed proteins in biological specimens .
antibodies ( r&d systems , minneapolis , minn ; pharmingen , san diego , calif ; biosource international , camarillo , calif ) were diluted to 0.5  mg / ml in pbs containing 0.05  mg / ml bsa , and 375  pl of each was spotted in quadruplicate onto the slides using a biochip arrayer ( packard biosciences , downers grove , ill ) .
each spot has a diameter of approximately 180  m with a center - to - center spacing of 270  m .
each slide was divided by a teflon mask into 16 subarrays , each with a diameter of 0.65  cm . within each subarray
, 256 spots were printed at known locations , containing 40 different features ( antibodies for specific antigens ) in quadruplicate ( 160 spots ) .
the remaining features comprised of controls for antibody immobilization , cy5-labeled mouse igg and internal calibrators ( biotin mouse igg ) . 

slides were blocked by adding 30  l blocking buffer ( 0.5% nonfat dry milk , 0.2% bsa , and 0.05% tween-20 in pbs ) to each subarray and incubating for 1 hour at 37c in a humidified chamber , and then washed by immersion in pbs containing 0.5% brij 35 for 2 minutes .
a 15  l aliquot of sample ( single analyte or mixture of multiple analytes ; serum , plasma , or culture supernatant ) was immediately added to each subarray , incubated for 30 minutes at 37c , and then washed as described above .
after analytes were captured , a 20  l aliquot of the appropriate mixture of biotinylated detection antibodies at 0.1  g / ml each was applied to individual arrays and incubated at 37c for 30 minutes in a humidified chamber .
slides were washed twice for two minutes in pbs containing 0.5% brij 35 . a mouse monoclonal antibiotin antibody - primer conjugate
was prepared by derivatization of a mouse monoclonal antibiotin igg ( jackson immunoresearch laboratories , inc , west grove , pa ) with a 5'-terminal amine - modified oligonucleotide primer , 5'-thiol - aaa aaa aaa aaa aaa cac agc tga gga tag gac at-3 ' , as previously described .
the conjugate was annealed with 75  nm circle 1 ( cyclic 5'-ctc agc tgt gta aca aca tga aga ttg tag gtc aga act cac ctg tta gaa act gtg aag atc gct tat tat gtc cta tc-3 ' ) in pbs containing 0.05% tween-20 and 1  mm edta at 37c for 30 minutes .
rca reaction mixture ( 20  l ) containing native t7 dna polymerase ( 20 u / ml ) , 1  mm dntps , ssdna - binding protein 22  g / ml , 1x sequenase buffer , 8% dmso , and 0.05  mm cy5 decorator ( 5'-cy5-tgt cct atc ctc agc tgg - cy5 ) was added to each subarray .
slides were incubated at 37c for 45 minutes , then washed twice in pbs containing 0.5% brij 35 and once in pbs , and spin - dried .
arrays were analyzed on an axon genepix 4000b scanner ( axon instruments inc , foster city , calif ) , and fluorescence quantitated by using genepix pro 3.0 quantitation software .
it consists of ( a ) analyte capture by antibody immobilized on microarray , ( b ) detection by biotinylated secondary antibody , ( c ) binding of antibiotin antibody - oligonucleotide conjugate pre - annealed with circle 1 , and ( d ) rolling - circle replication to generate long single - strand dna which is hybridized with oligonucleotide decorator .
this surface was chosen based on experiments indicating that cyanosilane - activated slides have the advantages of ( 1 ) simpler preparation , ( 2 ) less - expensive reagents , and ( 3 ) more uniformity of bound antibody spot morphology over thiolsilane - coated and cross - linker ( such as gmbs ) activated glass slides ( data not shown ) .
the concentration of capture antibody for printing was 
 optimized by comparing specific signal intensities from the same capture antibody printed on the chip at different concentrations chosen for microarray printing .
specific signals at 0.5  mg / ml reached the point of saturation ( data not shown ) , indicating that antibodies saturated the area available for immobilization in the spot .
the uniformity of antibody immobilization on the chip was assessed by measuring the coefficient of variation ( cv ) from the quadruplicate spots using cy5-labeled anti - isotype antibodies ( against the species used in the capture antibodies ) , previously reported as approximately 10% for each feature on the chip . rolling circle amplification enables amplification of signals tethered to proteins and nucleic acids [ 4 , 9 , 11 ] .
immunoassays incorporating rca have shown a large increase in sensitivity when compared with the direct assay .
the technique consists of protein capture by antibody immobilized on microarrays , detection by biotin - labeled second antibody specific for the captured protein , binding of a universal antibiotin antibody oligonucleotide conjugate pre - annealed with circle , and rca signal amplification where a long single - strand dna is generated by rolling - circle replication in the presence of dna polymerase and nucleotides , with hybridization of the rca product to fluorescent - labeled complementary oligonucleotide probes occurring simultaneously ( figure 1 ) . besides the signal amplification by rca ,
the sensitivity of the antibody array was further improved by two approaches : ( 1 ) choosing optimal capture and detection antibody pairs for the microarray immunoassay and ( 2 ) optimizing assay procedures .
optimization of antibodies from different sources ( a and b ) for choosing optimal pair for immunoassay on protein microarray with examples of ( a ) il-13 and ( b ) mcp-2 .
for individual features , if the matched antibody pair developed for conventional elisa was available , it was preferentially selected for the chip development . however , in terms of sensitivity and specificity , antibody pairs do not always function equivalently on plastic elisa plates and the glass - based microarrays .
antibody pairs from different manufacturers for the same antigen with similar sensitivity as elisa assays perform quite differently in microarrays .
thus it has become necessary to select optimal pairs for antibody microarrays by comparing all available antibodies and pairs . as an example , figure 2a shows the difference between two matched pairs of antibodies ( for elisa ) from different sources with specificity for detecting the cytokine il-13 using the antibody microarray .
the pair from source a showed strong signal intensity with a sensitivity of less than 100  pg / ml .
however , the pair from source b did not show signal even at concentrations as high as 5  ng / ml il-13 . selecting the antibody pair from source
a provided at least 50-fold increase of sensitivity when compared with the pair from source b. alternatively , the elisa antibody pair from the same set is not always the optimal for a microarray immunoassay .
neither of the elisa - matched pairs showed sensitivity better than 100  pg / ml on the microarray slides .
however , using a capture antibody from a and a detection antibody from b , the sensitivity for mcp-2 was improved to 10  pg / ml or better , a 10-fold increase in sensitivity relative to a single manufacturer 's reagents . for features without matched pairs ,
monoclonal antibodies with different binding epitopes or polyclonal antibodies were extensively evaluated for sensitivity ranges permitting detection of biologically relevant changes in protein expression .
cross - reactivity of capture antibodies and analytes ( ( a ) and ( c ) ) , capture antibodies and detection antibodies ( ( b ) and
( d ) ) on the subarrays 3 ( a ) and 4 ( b ) .
each subarray was printed with about 35 different capture antibodies ( listed on the chart ) .
cross - reactivity was determined by using different matched secondary antibody and 50  ng / ml or 0  ng / ml of its cognate analyte , which has been described in the text .
a shaded square off the diagonal indicates the presence of nonspecific signals ( intensity  300 ) .
none of these nonspecific signals exceed the intensity of 600 fluorescence intensity units ( images are scanned at pmt of 600 and power of 100% on axon scanner ) .
( a ) images of detecting 70 proteins in antibody microarray obtained with axon microarray scanner .
( b ) fluorescence intensities were derived from microarray images with averaging four spots of the same feature .
this involved optimizing detection antibody concentrations , conjugate and circle concentrations , incubation temperatures and time , and rca reaction solution composition .
analogous to sandwich elisa assays , the conditions giving the best signal to noise ratio were found to be 0.1  g / ml of detector with assay incubation time of 30 minutes at 37c .
titration of conjugate and circle pre - annealed to conjugate revealed the optimal concentration to be 2  g / ml conjugate with 75  nm circle .
other components , such as the concentration of single - strand dna binding protein ( ssb ) , were also optimized for maximum sensitivity in rca reaction mixtures ( figure 3 ) .
the analytical sensitivity of the microarray immunoassay was determined by using serial dilutions of each single purified antigen .
the sensitivity of detection , defined as the lowest concentration that delivered clear specific signal intensity above detection threshold ( mean intensity of the negative control plus 2 sd ) , was calculated .
sixty - five ( 43% ) of the 150 cytokine features had a sensitivity of  10  pg / ml , 52 ( 35% ) had a sensitivity of  100  pg / ml , 27 ( 18% ) had a sensitivity of  1000  pg / ml , and 6 ( 4% ) had a sensitivity of  1,000  pg / ml ( table 1 ) .
sixty - two of the cytokines represented on the chip are unique , with no corresponding commercially available elisa kits .
the sensitivity goal for these arrays was adequacy to detect biologically relevant ( ie , 2-fold ) increases in cytokine level above normal values in clinical fluids including serum and plasma .
although antibodies   are highly specific for their respective cognate proteins , it is possible that structurally related proteins might have similar epitopes . cross - reactivity or 
 nonspecific signals between immobilized capture antibodies and antigens or detection antibodies for 150 features were determined in three different ways .
first , the cross - reactivity of individual antigens or detection antibodies was determined by analyzing a single antigen at 0 and 50  ng / ml with its corresponding paired detection antibody .
any signals on the 0  ng / ml array were categorized as cross - reactivity between the detection and capture antibodies . with 50  ng / ml analyte , signals should have only been observed at the specific locations .
other signals after background subtraction ( the 0  ng / ml array ) were assumed to be cross - reactivity from the analyte .
nonspecific signals were eliminated by segregating all features into multiple subarrays , thus the capture antibodies producing nonspecific signals were physically separated from those particular detection antibodies or antigens .
this also allowed simultaneous detection of both components of a biological signaling system , such as a growth factor and its receptor .
residual cross - reactivity and nonspecific signals were first minimized by optimization of washing and blocking conditions .
next , a mixture of several analytes at concentrations of 10  ng / ml each was applied to the chip , followed by detection with the complete detection antibody cocktail .
signals were only observed where both the analyte and its specific detection antibody were present .
then a mixture of all analytes in the subarray at 10  ng / ml each was added to the microarray , but detection was with an incomplete cocktail of antibodies .
signals were only detected on those features corresponding to the specific detection antibodies added . as shown in figure 4 ,
both cross - reactivity between capture antibodies and analytes and cross - reactivity between capture antibodies and detection antibodies in each subarray were minimized .
approximately 99% of nonspecific binding pairs had fluorescence intensity units of less than 300 , used as the background signal minimum during the data quantitation .
interferences from biological specimen were screened using normal human serum and single detectors ( biotinylated detection antibody ) corresponding to each feature on the chip .
only specific signals of the proteins present in the specimen were detected by their associated detector antibody .
the antibody microarray was validated by running assays with normal human serum ( sigma , st .
signals corresponding to specific proteins were identified ( figure 5 ) , which are consistent with their presence with significant level in the normal human serum ( table 1 ) .
proteins with low fluorescence signal intensities agree with their very low level in serum ( if present ) , indicating that our antibody microarray provides a specific , robust , and high - throughput approach for global analysis of protein expression .
this surface was chosen based on experiments indicating that cyanosilane - activated slides have the advantages of ( 1 ) simpler preparation , ( 2 ) less - expensive reagents , and ( 3 ) more uniformity of bound antibody spot morphology over thiolsilane - coated and cross - linker ( such as gmbs ) activated glass slides ( data not shown ) .
the concentration of capture antibody for printing was 
 optimized by comparing specific signal intensities from the same capture antibody printed on the chip at different concentrations chosen for microarray printing .
specific signals at 0.5  mg / ml reached the point of saturation ( data not shown ) , indicating that antibodies saturated the area available for immobilization in the spot .
the uniformity of antibody immobilization on the chip was assessed by measuring the coefficient of variation ( cv ) from the quadruplicate spots using cy5-labeled anti - isotype antibodies ( against the species used in the capture antibodies ) , previously reported as approximately 10% for each feature on the chip .
rolling circle amplification enables amplification of signals tethered to proteins and nucleic acids [ 4 , 9 , 11 ] .
immunoassays incorporating rca have shown a large increase in sensitivity when compared with the direct assay .
the technique consists of protein capture by antibody immobilized on microarrays , detection by biotin - labeled second antibody specific for the captured protein , binding of a universal antibiotin antibody oligonucleotide conjugate pre - annealed with circle , and rca signal amplification where a long single - strand dna is generated by rolling - circle replication in the presence of dna polymerase and nucleotides , with hybridization of the rca product to fluorescent - labeled complementary oligonucleotide probes occurring simultaneously ( figure 1 ) . besides the signal amplification by rca ,
the sensitivity of the antibody array was further improved by two approaches : ( 1 ) choosing optimal capture and detection antibody pairs for the microarray immunoassay and ( 2 ) optimizing assay procedures .
optimization of antibodies from different sources ( a and b ) for choosing optimal pair for immunoassay on protein microarray with examples of ( a ) il-13 and ( b ) mcp-2 .
for individual features , if the matched antibody pair developed for conventional elisa was available , it was preferentially selected for the chip development .
however , in terms of sensitivity and specificity , antibody pairs do not always function equivalently on plastic elisa plates and the glass - based microarrays .
antibody pairs from different manufacturers for the same antigen with similar sensitivity as elisa assays perform quite differently in microarrays .
thus it has become necessary to select optimal pairs for antibody microarrays by comparing all available antibodies and pairs . as an example , figure 2a shows the difference between two matched pairs of antibodies ( for elisa ) from different sources with specificity for detecting the cytokine il-13 using the antibody microarray .
the pair from source a showed strong signal intensity with a sensitivity of less than 100  pg / ml .
however , the pair from source b did not show signal even at concentrations as high as 5  ng / ml il-13 . selecting the antibody pair from source
a provided at least 50-fold increase of sensitivity when compared with the pair from source b. alternatively , the elisa antibody pair from the same set is not always the optimal for a microarray immunoassay .
neither of the elisa - matched pairs showed sensitivity better than 100  pg / ml on the microarray slides .
however , using a capture antibody from a and a detection antibody from b , the sensitivity for mcp-2 was improved to 10  pg / ml or better , a 10-fold increase in sensitivity relative to a single manufacturer 's reagents . for features without matched pairs ,
monoclonal antibodies with different binding epitopes or polyclonal antibodies were extensively evaluated for sensitivity ranges permitting detection of biologically relevant changes in protein expression .
cross - reactivity of capture antibodies and analytes ( ( a ) and ( c ) ) , capture antibodies and detection antibodies ( ( b ) and ( d ) ) on the subarrays 3 ( a ) and 4 ( b ) .
each subarray was printed with about 35 different capture antibodies ( listed on the chart ) .
cross - reactivity was determined by using different matched secondary antibody and 50  ng / ml or 0  ng / ml of its cognate analyte , which has been described in the text .
a shaded square off the diagonal indicates the presence of nonspecific signals ( intensity  300 ) .
none of these nonspecific signals exceed the intensity of 600 fluorescence intensity units ( images are scanned at pmt of 600 and power of 100% on axon scanner ) .
( a ) images of detecting 70 proteins in antibody microarray obtained with axon microarray scanner .
( b ) fluorescence intensities were derived from microarray images with averaging four spots of the same feature .
the second approach of improving sensitivity was to optimize the assay procedures . this involved optimizing detection antibody concentrations , conjugate and circle concentrations , incubation temperatures and time , and rca reaction solution composition .
analogous to sandwich elisa assays , the conditions giving the best signal to noise ratio were found to be 0.1  g / ml of detector with assay incubation time of 30 minutes at 37c .
titration of conjugate and circle pre - annealed to conjugate revealed the optimal concentration to be 2  g / ml conjugate with 75  nm circle .
other components , such as the concentration of single - strand dna binding protein ( ssb ) , were also optimized for maximum sensitivity in rca reaction mixtures ( figure 3 ) .
the analytical sensitivity of the microarray immunoassay was determined by using serial dilutions of each single purified antigen .
the sensitivity of detection , defined as the lowest concentration that delivered clear specific signal intensity above detection threshold ( mean intensity of the negative control plus 2 sd ) , was calculated .
sixty - five ( 43% ) of the 150 cytokine features had a sensitivity of  10  pg / ml , 52 ( 35% ) had a sensitivity of  100  pg / ml , 27 ( 18% ) had a sensitivity of  1000  pg / ml , and 6 ( 4% ) had a sensitivity of  1,000  pg / ml ( table 1 ) .
sixty - two of the cytokines represented on the chip are unique , with no corresponding commercially available elisa kits .
the sensitivity goal for these arrays was adequacy to detect biologically relevant ( ie , 2-fold ) increases in cytokine level above normal values in clinical fluids including serum and plasma .
although antibodies   are highly specific for their respective cognate proteins , it is possible that structurally related proteins might have similar epitopes . cross - reactivity or 
 nonspecific signals between immobilized capture antibodies and antigens or detection antibodies for 150 features were determined in three different ways .
first , the cross - reactivity of individual antigens or detection antibodies was determined by analyzing a single antigen at 0 and 50  ng / ml with its corresponding paired detection antibody .
any signals on the 0  ng / ml array were categorized as cross - reactivity between the detection and capture antibodies . with 50  ng / ml analyte , signals should have only been observed at the specific locations .
other signals after background subtraction ( the 0  ng / ml array ) were assumed to be cross - reactivity from the analyte .
nonspecific signals were eliminated by segregating all features into multiple subarrays , thus the capture antibodies producing nonspecific signals were physically separated from those particular detection antibodies or antigens .
this also allowed simultaneous detection of both components of a biological signaling system , such as a growth factor and its receptor .
residual cross - reactivity and nonspecific signals were first minimized by optimization of washing and blocking conditions .
next , a mixture of several analytes at concentrations of 10  ng / ml each was applied to the chip , followed by detection with the complete detection antibody cocktail .
. then a mixture of all analytes in the subarray at 10  ng / ml each was added to the microarray , but detection was with an incomplete cocktail of antibodies .
as shown in figure 4 , both cross - reactivity between capture antibodies and analytes and cross - reactivity between capture antibodies and detection antibodies in each subarray were minimized .
approximately 99% of nonspecific binding pairs had fluorescence intensity units of less than 300 , used as the background signal minimum during the data quantitation .
interferences from biological specimen were screened using normal human serum and single detectors ( biotinylated detection antibody ) corresponding to each feature on the chip .
only specific signals of the proteins present in the specimen were detected by their associated detector antibody .
the antibody microarray was validated by running assays with normal human serum ( sigma , st .
signals corresponding to specific proteins were identified ( figure 5 ) , which are consistent with their presence with significant level in the normal human serum ( table 1 ) .
proteins with low fluorescence signal intensities agree with their very low level in serum ( if present ) , indicating that our antibody microarray provides a specific , robust , and high - throughput approach for global analysis of protein expression .
immunoassays on microarrays hold appeal for studies requiring the ability to quantify many selected proteins simultaneously . considerable progress has been made in this area recently in terms of increased assay sensitivity and complexity ( ie , degree of multiplexing ) .
rolling - circle amplification facilitates the use of such arrays with its powerful degree of signal enhancement and simultaneous detection of a large number of defined analytes .
such robust systems will enable routine use of antibody arrays in both research and diagnostic modalities .
 protein microarray - based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions . 
 the development of high - throughput , sensitive , convenient , and cost - effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes . in this paper , we describe the generation of highly multiplexed , antibody - based , specific , and sensitive protein microarrays coupled with rolling - circle signal amplification ( rca ) technology . 
 a total of 150 cytokines were simultaneously detected in an rca sandwich immunoassay format . 
 greater than half of these proteins have detection sensitivities in the pg / ml range . 
 the validation of antibody microarray with human serum indicated that rca - based protein microarrays are a powerful tool for high - throughput analysis of protein expression and molecular diagnostics . 