A water-stable protected isocyanate glass array substrate

ANALYTICAL

Analytical Biochemistry 326 (2004) 55–68

BIOCHEMISTRY

www.elsevier.com/locate/yabio

A water-stable protected isocyanate glass array substrate

Seshi R. Sompuram,a,b,* Kodela Vani,a Lan Wei,c Halasya Ramanathan,a Sarah Olken,a

and Steven A. Bogena,b

a Medical Discovery Partners LLC, 715 Albany St., Boston, MA 02118, USAb Department of Pathology and Laboratory Medicine, Boston University School of Medicine, 715 Albany St., Boston, MA 02118, USA

c Expression Array Core Facility, Tufts Center for Vision Research, Tufts University School of Medicine, Boston, MA 02111, USA

Received 5 September 2003

Abstract

We describe the performance of a new glass attachment chemistry for arrays that is particularly well suited to attachment of small

molecules, such as peptides. The attachment chemistry is a protected isocyanate (PI) group. Isocyanate groups are well suited to

serving as a glass coating for arrays, in that they are highly reactive with many different types of biological compounds. However,

they are generally so reactive as to be unstable. The new feature of the PI slide coating is its stability. It can withstand immersion in

water without loss of reactivity and has at least a 1-year shelf life. The high reactivity of the PI group results in a rapid coupling

reaction (<15min) and is particularly useful for attaching small molecules, such as peptides. Since isocyanates bind to both amines

(forming a urea linkage) and hydroxyl groups (forming a carbamate bond), we tested the ability of the PI coating to bind to a wide

variety of compounds. We found that the PI slide coating can directly attach to peptides, proteins, carbohydrates, lipooligosac-

charides, and DNA. The sensitivity of detection for these compounds is comparable to that of other previously published array

substrates.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Isocyanate; Microarrays; Glass slide coating; Diagnostics

The microarray has emerged as a preferred multi-

plexed screening format because of its high throughput,

density, sensitivity, and specificity. One of the challenges

in printing arrays relates to the method used for im-

mobilization of the macromolecule. A wide variety of

methods for immobilization of macromolecules, in-cluding both direct synthesis of DNA oligonucleotides

onto the microarray [1] and attachment of preformed

DNA or proteins, have been developed. For the latter,

attachment of preformed macromolecules to a glass

surface, there are several different options to choose

from. Glass attachment chemistries can be divided into

four general categories: covalent, noncovalent, diffusion,

and affinity-based attachment [2]. Each category has itsmerits and drawbacks.

For a separate project, we required a reliable method

for avidly attaching small peptides to a glass surface

[3,4]. Moreover, the method needed to provide a stable

* Corresponding author. Fax: 1-617-638-4333.

E-mail address: [email protected] (S.R. Sompuram).

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2003.11.008

covalent linkage to glass that will not be disrupted by

organic solvents or high temperatures. The glass coating

also needed to be simple and inexpensive to manufac-

ture. Existing methods of immobilization onto glass did

not meet these requirements. These needs caused us to

revisit isocyanate glass coupling chemistries and developa modification that renders it practical for array pur-

poses.

Affinity-based methods of protein or DNA immobi-

lization include nickel- [5] or avidin-coated [6–8] glass

slides. Both methods are reported to provide a strong,

specific attachment with a low level of background

fluorescence. However, the moiety to be immobilized

must be covalently modified to bear a polyhistidine orbiotin affinity tag. That requirement burdens the sample

preparation step and limits the range of analytes that

can be bound to glass.

Aminosilane and poly-LL-lysine coatings are popular

noncovalent methods of attaching DNA or proteins to

glass [9–12]. These coatings do not require modification

of the DNA or protein and are widely used. However,

mail to: [email protected]

56 S.R. Sompuram et al. / Analytical Biochemistry 326 (2004) 55–68

they tend to be suitable only for larger molecules. Pre-sumably, larger molecules bear a sufficient negative

charge so as to be electrostatically attracted to the

positively charged, protonated amine. Small oligonu-

cleotides or peptides do not attach well to these posi-

tively charged surfaces.

Because of the limitations associated with noncova-

lent glass attachment chemistries, we then considered

the available covalent glass immobilization methods.These include epoxide-, isothiocyanate-, N -hydroxysuc-

cinimide (NHS)1 ester-, amine-, semicarbazide-, and

aldehyde-derivatized surfaces [2,5,13–16]. Covalent at-

tachment provides for a more durable and stable linkage

to the glass substrate, capable of withstanding high

temperatures or organic solvents. However, preactivated

covalent attachment chemistries reportedly suffer from

several disadvantages. First, the molecule that is to beattached must have the appropriate reactive group. For

example, to use epoxides or succinimide esters for nu-

cleic acids, oligonucleotides must be modified with a

terminal amine group [17]. Semicarbazide-coated glass

requires that the moiety be derivatized with a benzal-

dehyde group. These requirements for premodification

of the sample burdens the sample preparation step and

can limit the types of materials that can be attached.In addition, many of the covalent immobilization

methods are not optimal for small molecules, such as

peptides. For example, our (unpublished) experience

was that peptides do not avidly cross-link to aldehyde-

coated slides. More reactive groups such as epoxides

or N -hydroxysuccinimide esters are better for immo-

bilization of small molecules, but they are less stable

[16].Isocyanates potentially represent an ideal covalent

DNA or protein immobilization chemistry, in that they

can react with many functional groups including amines,

hydroxyls, and carboxyl groups. Such a wide diversity of

potential chemical reactivity renders them highly ver-

satile, since these groups are found in nucleic acids,

proteins, and carbohydrates. Moreover, the isocyanate�shigh degree of chemical reactivity lends itself well to thecovalent immobilization of small molecules, such as

short oligonucleotides or peptides. The problem with

isocyanate immobilization chemistries is that their high

reactivity is also associated with chemical instability and

poor shelf life characteristics. Namely, the isocyanate

will readily combine with water, leaving it unreactive

and unable to immobilize DNA or proteins. Such re-

activity to water makes it sensitive to the humidity in air.

1 Abbreviations used: NHS, N -hydroxysuccinimide; PI, protected

isocyanate; ER, estrogen receptor; LOS, lipooligosaccharides; PBS,

phosphate-buffered saline; RT, room temperature; HRP, horseradish

peroxidase; SSC, sodium chloride and sodium citrate; M-MLV,

molony murine leukemia virus; DAB, 3,3-diaminobenzidene; IHC,

immunohistochemical; PR, progesterone receptor.

Presumably because of these difficulties, there are nocommercial vendors to our knowledge offering isocya-

nate-derivatized glass slides for spotting arrays.

This new type of isocyanate coupling chemistry

overcomes the stability problem. As a result, this at-

tachment chemistry appears to have the benefits of a

highly reactive chemical group without the concomitant

drawbacks. Namely, we describe in this paper the ver-

satility and stability of a protected isocyanate (PI)-de-rivatized microscope glass slide surface. The PI coating

appears to be particularly advantageous in immobilizing

small molecules such as peptides. Furthermore, we

demonstrate that the coupling reaction is rapid, occur-

ring within minutes. We propose that this glass

attachment chemistry represents a general purpose im-

mobilization chemistry to microscope glass slides and is

uniquely well suited to attaching small molecules, suchas oligonucleotides, peptides, or oligosaccharides.

Materials and methods

Analytes for coupling to slides

Peptide Pep 3ER1D5 has been previously described[3]. It was synthesized by SynPep Corp. (Dublin, CA).

Recombinant estrogen receptor (ER) a protein was

purchased from Panvera Corp. (Madison, WI). Poly-

clonal mouse IgG was purchased from Sigma Chemical

Co. (St. Louis, MO) or the Pierce Chemical Co.

(Rockford, IL). Two lipooligosaccharides (LOS) that

were extracted from Nesseria gonnohorea strains 1523 or

WG were the kind gift of Dr. S. Gulati (Maxwell Fin-land Laboratory of Infectious Diseases, Boston Uni-

versity School of Medicine, Boston, MA). Dextran

(a1 ! 6) was the kind gift of Dr. J. Sharon (Department

of Pathology and Laboratory Medicine, Boston Uni-

versity School of Medicine). Aminobiotin (biotin PEO-

LC-amine) was purchased from Pierce Chemical Co.

(Rockford, IL). For non-DNA work, poly-LL-lysine

slides (Silane-Prep slides) were purchased from SigmaChemical Co., whereas aminosilane slides (Superfrost

Plus) were purchased from VWR (West Chester, PA).

Primary antibodies for detection of analytes

The 1D5 mAb [18] binds to both recombinant ER aand to a synthetic cyclic peptide termed Pep 3ER1D5

[3]. The 1D5 mAb was purchased from DakoCytoma-tion (Carpinteria, CA) and was used at �3 lg/ml, unless

otherwise indicated. Lipooligosaccharide was detected

with mAb 2C7, a gift from Dr. S. Gulati (Boston Uni-

versity School of Medicine). It was used at a concen-

tration of 10 lg/ml in PBS/0.05% Tween 20. The

dextran-specific mAbs 26.4.1, 298.1.3, and 255.1.1B

were the kind gift of Dr. J. Sharon (Boston University

S.R. Sompuram et al. / Analytical Biochemistry 326 (2004) 55–68 57

School of Medicine). These murine mAbs were detectedwith anti-mouse IgG–biotin conjugate, purchased from

Vector Laboratories (Burlingame, CA).

Protected isocyanate slide coating

The PI slides have been previously described in

publications relating to their application for immuno-

histochemistry quality control and for retention ofpoorly adherent histologic tissue sections [3,4,19]. The

PI coating is applied to plain microscope slides, pur-

chased from VWR International. Their manufacture is

accomplished in two steps. First, the slides are placed

into slide racks and immersed into an aminosilane so-

lution. Subsequently, the pendant amine group of the

aminosilane is converted into an isocyanate moiety by

the addition of a phosgene equivalent. The phosgeneequivalent also has a blocking group, creating a pro-

tected isocyanate moiety due to the blocking group. The

slides are then stored in the refrigerator (4–7 �C) until

they are used. To couple any of the analytes used in this

paper, the analyte was dissolved in 500mM KPO4

buffer, pH 8.9, and 1 ll of each dilution was spotted

onto PI-coated slides. The isocyanate slides were

warmed on a hot plate, to 42–45 �C, to accelerate thecoupling process. After coupling, the slides are then

usually blocked with 0.1% Immunoassay Post-Coating

Buffer (US Biological, Swampscott, MA), 0.025% Bo-

vine Gamma Globulins (Sigma Chemical Co.) for

15min at room temperature (RT), dissolved in PBS, to

quench unreacted PI sites on the glass.

Carbohydrate coupling and detection

LOS and dextran were diluted either in KPO4 (0.5M,

pH �8.9) or boric acid (0.2M, pH 6.0) and applied to

the PI slide as 1-ll spots. The coupling of LOS or dex-

tran to the PI-coated glass slide was facilitated by in-

cubating the slides for 15min at 42–45 �C. Dextran spots

were subsequently detected by incubation with one of

the dextran-specific mAbs for 30min at RT. LOS spotswere detected with mAb 2C7, 30min at RT. For both

dextran and LOS detection, the primary mAbs were

detected by an immunoenzymatic technique, using an

anti-mouse immunoglobulin reagent conjugated to ei-

ther horseradish peroxidase (HRP) or alkaline phos-

phatase (both from the Sigma Chemical Co).

Nucleic acid arraying onto PI slides

Purified PCR products of 768 mouse cDNA clones

were resuspended in 3� sodium chloride and sodium

citrate buffer (3� SSC) with 1.5M Betaine (Sigma) to

give a final concentration around 150 ng/ll. The DNA

fragments above were arrayed in duplicates by Micro-

Grid II arrayer (Apogent Discoveries, Hudson, NH) in

4� 2 subarrays of 14 rows and 14 columns each. Thespot-to-spot space was 280 lm. After arraying, slides

were UV cross-linked and baked at 80 �C for 2 h. The PI

slides do not need UV cross-linking, but it is not detri-

mental and the comparison slides (on poly-LL-lysine and

aminosilane slide coatings) were so treated. Thus, we

treated all the slides equivalently after printing. Before

hybridization, slides were immersed in boiling water for

3min and then dried by spinning in a Beckman GS-15Rcentrifuge at 1000 rpm for 1min.

Probe labeling

The cDNA probe was generated from mouse brain

RNA samples that were extracted, reverse transcribed,

and then labeled with Cy5 with Cyscribe Post-labeling

kit (Amersham Biosciences, Piscataway, NJ). The first-strand cDNA reverse transcript was labeled with amino

allyl-dUTP from 10 lg total RNA using the oligo(dT)

primer and molony murine leukemia virus (M-MLV)

reverse transcriptase (Invitrogen, Carlsbad, CA). After

purification with Cyscribe GFX Purification Kit

(Amersham Biosciences), the labeled cDNA was reacted

with CyDye NHS esters to incorporate Cy5. The cDNA

was separated from the uncoupled CyDye NHS estersby a second purification with Cyscribe GFX Purification

Kit. Cy5-labeled cDNA was dried in DNA110 Speed-

Vac concentrator (Thermo Savant, Holbrook, NY). The

pellet was resuspended in 60 ll 1� hybridization buffer

(Cyscribe GFX purification kit, Amersham Biosciences)

with 25% formamide and 30 ll was used for hybridiza-

tion. The rest was stored at )20 �C.

Hybridization

The hybridization solution tube was immersed for

3min in boiling water and then 30 ll was applied to the

PI-coated slide array. The slide was covered with a

22� 22-mm cover slip. The slide was incubated in a

Hybridization Chamber (Corning, Kennebunk, ME) at

42 �C, overnight.

Scanning of arrays

Nucleic acid array slides were washed once with 1�SSC, 0.2% SDS for 10min, twice with 0.1� SSC, 0.2%

SDS for 10min, and twice with 0.1� SSC alone for

1min. The slides were then spun-dry in a Beckman GS-

15R centrifuge at 1000 rpm for 1min. The slides werethen scanned in a ScanArray 4000 scanner (PerkinElmer

Life Sciences, Boston, MA).

Immunohistochemical detection

In order to detect primary antibodies that are bound

to an analyte on the PI slide surface, a standard labeled


streptavidin biotin detection system was used, accord-ing to a previously published protocol [3,4]. Briefly,

these steps include incubation with biotin-conjugated

horse anti-mouse IgG (heavy and light chain-specific)

secondary antibody (Vector Laboratories) for 20min at

RT. The slides were then rinsed in PBS and incubated

with HRP-conjugated streptavidin for 20min at RT.

After rinsing off unbound enzyme conjugate, the anti-

body–enzyme complex was then visualized with liquid-stable 3,30-diaminobenzidine (DAB) tetrahydrochloride

(2mg/ml, Sigma)/0.02% hydrogen peroxide (H2O2) for

10min, and subsequently enhanced with 5% (w/v)

copper enhancer (cupric sulfate pentahydrate) for

10min.

We also measured the relative quantity of certain

analytes bound to the PI glass slide without the use of

primary antibodies. Aminobiotin was coupled to thePI-coated glass surface, to demonstrate the ability of

the PI slide to couple to low-molecular-weight com-

pounds (Fig. 2A). It was detected by incubating the

slides with streptavidin–horseradish peroxidase conju-

gate (Vector Laboratories) for 10min at RT. Also

shown in Fig. 2A is the relative sensitivity of the cou-

pling of streptavidin to the PI-coated glass surface. The

relative quantity of streptavidin was quantified by in-cubating the glass slide with 5 lg/ml biotin-conjugated

horseradish peroxidase (Sigma; Pierce) for 20min at

RT. In each instance, the quantity of the detecting

moiety (a horseradish peroxidase conjugate) was then

measured by incubating the glass slides with DAB/

H2O2 for 10min at RT.

Spot color quantification

Spots were approximately 3mm in diameter and

were colored brown after incubation with DAB/H2O2.

The colorimetric intensity of these immunoenzymati-

cally stained peptide, protein, carbohydrate, or lipo-

oligosaccharide spots was measured by scanning the

slides with a flat bed scanner (Perfection 1200U, Ep-

son America, Inc., Torrance, CA), set at gray scalewith a resolution of 600 dpi. The image was stored in

Adobe Photoshop. The color intensity of the spots was

then quantified using a Scion image program (Scion

Corp., Frederick, MD). Peptide spot color intensity is

expressed as mean pixel optical density, on a 1–256

scale.

Stability of isocyanate-coated slides

The shelf life of PI-coated slides was tested by mea-

suring their efficacy in coupling molecules. PI-coated

slides were stored at 4 �C before use. Slides were tested

at various time points for their ability to couple peptide

Pep 3ER1D5. A freshly coated slide was used as a po-

sitive control.

Stability of peptides or proteins coupled to isocyanate-

coated slides

The shelf life of peptides coupled to PI-coated slides

was tested over a 2-year time period. PI-coated slides

were coupled with Pep 3ER1D5 peptide, as described

above. The slides were stored at 4 �C. At various time

points afterward, the analytes on the PI-coated slides

were detected using an immunohistochemical (IHC)procedure. As an assay control, peptide was coupled to

freshly coated (less than 7 days old) PI slides and stained

along with test slides.

Stripping and reapplication of antibodies from the PI-

coated glass surface

This protocol is described in a flow chart (Fig. 7A).To test the strength of the bond that couples analytes to

the PI-coated glass surface, we attempted to elute off

recombinant ER a protein using one of two stripping

buffers. The recombinant ER a protein is detected with

the 1D5 ER mAb. We used two stripping buffers, either

a citrate buffer (8.1mM sodium citrate and 1.97mM

citric acid, pH 6.0) or an ‘‘ECL’’ stripping buffer

(62.5mM Tris, pH 6.7, 2% SDS, 100mM b-mercap-toethanol). Slides stripped in the citrate buffer were

boiled in a pressure cooker (Nordic Ware, Minneapolis,

MN), which itself was in a microwave oven set on the

‘‘high’’ setting for 20min. Slides stripped in the ECL

buffer were incubated at 50 �C for 30min. Stripped slides

were then rinsed by immersing the slides five times in

PBS.

As a positive control, a group of slides bearing rERprotein was not treated with a stripping solution. This

group represents the maximal intensity signal that can

be obtained using the IHC detection protocol using the

ER 1D5 mAb, followed by biotin-conjugated anti-

mouse IgG (secondary), streptavidin–HRP (tertiary),

and finally the DAB/H2O2 substrate. Spot intensity was

measured as described earlier.

The negative control group of slides was designed totest whether the stripping buffers could, in fact, elute

noncovalently bound proteins. Since antibodies bind

noncovalently, we measured whether the 1D5 mAb was,

in fact, eluted after treatment. In this negative control

group, rER protein-spotted slides were incubated with

the 1D5 mAb. The slides were then treated with one of

the elution buffers. We then measured the amount of

1D5 mAb remaining on the glass slides after elutionwith a stripping buffer. To do so, we incubated the slide

with an anti-mouse IgG–biotin conjugate, followed by a

streptavidin–HRP conjugate, as described previously.

The experimental group was processed like the neg-

ative control group, up to the point where the 1D5 mAb

is eluted off. For the experimental group, we wished to

ascertain whether the rER protein was also eluted off the


slide. Therefore, we reapplied the 1D5 mAb, for a sec-ond incubation. If the rER protein was not eluted, then

the 1D5 ER mAb will bind to it. The presence of the

1D5 ER mAb on the slides was then detected using a

standard labeled streptavidin–biotin immunohisto-

chemical detection protocol, for relative quantification

of the amount of rER protein on the glass slide.

Results

Protected isocyanate glass slide coating

Isocyanate groups are well known in organic chem-

istry as highly reactive groups [20]. Although isocyanate

chemistry on glass has been described [21], it has not

been demonstrated to be practical for bioassays such asarrays. The challenge with isocyanates is that they are so

strongly reactive that they will even react with water,

rendering them susceptible to moisture in the air. For

this reason, many descriptions of isocyanate reactions

required that the moiety be dissolved in a nonaqueous

organic solvent. This feature makes the isocyanate

chemistry unstable and unsuitable as a preactivated

substrate for bioassays. The use of such organic solventswould further limit coupling of biological molecules, as

they would often be denatured in organic solvents.

To address the stability need and thereby benefit from

the isocyanate group�s broad range of chemical reac-

tivities to many different types of biological molecules,

we developed a method to synthesize an isocyanate with

a chemical protecting group. Fig. 1 schematically illus-

trates the unique feature of the isocyanate glass activa-tion chemistry. The protecting group is not displaced by

water, preserving the chemical reactivity of the isocya-

nate in an aqueous environment. This makes the reac-

tive group insensitive to moisture, an advantageous

characteristic of our PI glass activation chemistry.

Amine groups on peptides or proteins, however, are

Fig. 1. Chemical reactivity of the protected isocyanate (PI)

capable of displacing the protecting group and forminga covalent urea linkage. Hydroxyl groups, such as those

on carbohydrates and nucleic acids, or carboxyl groups

on proteins and peptides are also capable of displacing

the protecting group, forming a carbamate bond. These

reactions are illustrated in Fig. 1.

Detection sensitivity for varying-sized molecules

Previous reports describing array sensitivities have

examined the issue in two different contexts. For some,

the detection sensitivity for arrays was reported as the

threshold of detection for an analyte on a glass-immo-

bilized spot [22]. The sensitivity was the lowest detect-

able amount of material per spot. Other reports describe

sensitivity in an immunoassay context. Namely, the

sensitivity was described as the lowest concentration of amolecule in solution that could be detected by an array

[23–25]. In this context, the arrays are composed of

antibodies or antigens. We report our data on both of

these parameters. In Figs. 2A and B, we describe the

amount of analyte that can be detected per microliter

spot. In Fig. 3, we describe the concentration of anti-

body in solution that can be detected.

Fig. 2A demonstrates the detection sensitivity ofglass-immobilized analytes for PI slides, using conven-

tional immunoenzymatic detection techniques (de-

scribed under Materials and methods). In this series of

experiments, 1 ll droplets containing analytes of varying

molecular weights were applied to the protected isocy-

anate glass coating. The droplets form 3-mm-diameter

spots. Our particular application required spots that

could be seen without the aid of a microscope [3,4]. Thedata, shown in Fig. 2A, demonstrate that the detection

limit is approximately in the nanogram per microliter

spot range, depending upon the analyte. Since a typical

microarray uses nanoliter (rather than microliter) spots,

the absolute amount of analyte would be proportion-

ately less in a nanoliter-sized spot.

slide coating, forming urea and carbamate linkages.

Fig. 2. (A) Detection limit for analytes of varying molecular weights.

The data illustrate the colorimetric intensity (y axis) of four different

analytes that were applied to the PI slide coating, after immunoenzy-

matic detection. The concentration of the analyte applied to the slide

(in a 1-ll droplet) is shown on the x axis. In these experiments, we used

1-ll spots of 3mm diameter. The analyte spots are developed by im-

munohistochemical methods appropriate for the analyte. The colori-

metric signal intensity of the spots is subtracted from the blank or

negative control spots and is plotted on the y axis. The mean blank, or

negative control, value of the glass alone was 24.7 with a standard

error (SE) of �0.5. The data illustrate that the PI slide coating works

well not only for large analytes but also for low-molecular-weight

analytes. (B) Comparison of detection sensitivities between PI, poly-LL-

lysine, and aminosilane slide coatings for a small peptide (left) and a

protein (right). The peptide has a m.w. of 2370Da. The protein,

murine IgG, has a m.w. of approximately 150 kDa. After immunode-

tection of the glass-bound peptide or protein, the resulting colorimetric

signal intensity is plotted against the y axis.

Fig. 3. Sensitivity and dynamic range for measuring antibodies in so-

lution, using the PI-coated glass array substrate. The ER 1D5 mAb

binds to a peptide mimetic that resembles a portion of the estrogen

receptors. The PR 636 mAb binds to a peptide mimetic that resembles

a portion of the progesterone receptor. We coupled 0.5 pmol ER

peptide and 10 pmol PR peptide to the PI slide at 42 �C. The x axis

indicates the concentrations of each antibody. The resulting colori-

metric signal for each antibody concentration is represented on the yaxis. Each symbol represents the mean�SE of triplicate measurements

on the same slide.


The analytes included (in descending molecular

weight order) polyclonal mouse IgG (m.w. 150 kDa),

streptavidin (m.w. 53 kDa), Pep 3ER1D5 (m.w.

2370Da), and aminobiotin (m.w. 419Da). Pep 3ER1D5

is a 19-amino-acid-long peptide that binds to the antigenbinding site of the 1D5 estrogen receptor antibody. This

feature facilitates detection of the peptide. This peptide

and its affinity to the 1D5 estrogen receptor antibody

have been previously described [3]. The concentrations

for each are as indicated on the x axis in Fig. 2A. Each

molecule was detected by immunoenzymatic methods,

appropriate for the molecule (see Materials and meth-

ods). The different types of molecules, by necessity, re-quired different detection protocols. The degree of color

intensity and sensitivity varies somewhat between the

different analytes, presumably because of the different

detection methods and their relative levels of amplifi-

cation. Although the affinity constant of target–ligand

pairs can also affect the apparent detection sensitivity

limit, the affinity is probably a small issue compared to

the differences in the detection systems. Despite the

variability in detection thresholds among the differenttypes of compounds, the general result in Fig. 2A is that

the level of detection sensitivity for each approaches

approximately 1 ng/ll spot (or 1 pg/nl spot, as expressedfor a microarray context). This range is comparable to

other slide coatings when comparable immunoenzy-

matic detection methods are used (see Discussion).

Fig. 2B demonstrates a direct comparison of the de-

tection sensitivities of PI-, aminosilane-, and poly-LL-ly-sine-coated slides for a small peptide (m.w. 2370Da)

and a globular protein (IgG, m.w. 150 kDa). The con-

centrations of the peptide (Fig. 2B, left) or protein

(Fig. 2B, right) that are coupled to various slide coatings

are illustrated on the x axis. The glass-bound peptide or

protein was detected with an appropriate specific anti-

body followed by a labeled streptavidin–biotin detection

protocol (Materials and methods). The colorimetricsignal intensity for each concentration of peptide (left)

or protein (right) is shown on the y axis. The data

suggest that small peptides can couple efficiently to the

PI slide coating and hence give superior sensitivity

compared to the poly-LL-lysine and aminosilane slides. In

general, all three slides display reasonable performance

for a large protein (Fig. 2B, right), but the PI slide still

displays a slightly superior sensitivity over the other twoslide coatings.

Fig. 3 shows the detection sensitivity for antibodies in

solution using an immunoenzymatic detection system

(Materials and methods). This would be analogous

to a serologic assay, measuring the lowest antibody


concentration that can still be detected. As a capturereagent on the glass, we use peptides that are antibody

specific. These peptides simulate the antigen to which

the antibodies normally bind. These peptides were iso-

lated using phage display and have been previously de-

scribed [3,4]. The 1D5 estrogen receptor antibody binds

to an ER mimetic peptide whereas the 636 progesterone

receptor (PR) antibody binds to a separate PR mimetic

peptide. We observed a broad dynamic assay range forboth peptides, as shown in Fig. 2B, with the lower limit

of sensitivity around the lowest concentration point

tested (37 ng/ml).

Rapid binding kinetics

Fig. 4 represents the binding kinetics of the Pep

3ER1D5 peptide to the PI slide coating. The relativeamount of peptide that attached to the PI-coated glass is

expressed as the spot intensity (y axis), i.e., the mean

pixel intensity on a scale of 1–256. In this experiment,

1 ll spots containing 0.5 pmol of Pep 3ER1D5 peptide

were placed on replicate PI-coated glass slides and in-

cubated at the indicated temperatures (RT, 30, 40, or

50 �C). The reaction was then stopped after 1, 5, 15, or

Fig. 4. Kinetics of peptide coupling to PI-coated slides at different

temperatures. We coupled 0.5 pmol ER peptide at various tempera-

tures and time intervals. Each symbol represents the mean�SE of

triplicate measurements on the same slide.

Fig. 5. (A) Stability of PI-coated slides in water. Before coupling the ER pepti

not immersed (left bar) in water. The data illustrate that the PI slide coating is

triplicate measurements on the same slide. (B) Shelf life stability of PI slides. T

3ER1D5 (0.5 pmol) peptide was tested at the times indicated on the x axis. Th

30min by rinsing the slide with distilled water. Theunreacted PI groups were then quenched by incubation

with a protein blocking solution, as described under

Materials and methods. The Pep 3ER1D5 was then

detected by a standard immunohistochemical procedure

using the 1D5 estrogen receptor antibody, as described

under Materials and methods. Colorimetric intensity of

the resultant spots are plotted on the y axis of Fig. 4.

The data demonstrate that the Pep 3ER1D5 binds toPI-coated glass slides better and faster at elevated tem-

peratures. Regardless of the temperature, the reaction is

complete in 5–15min. This time compares quite favor-

ably to other covalent coupling chemistries, which gen-

erally require several hours or even overnight (see

Discussion).

The PI slide coating is water-stable

PI-coated slides were tested for sensitivity to water by

immersing the slides in distilled water at room temper-

ature for 1 h. A control set of PI-coated slides was not

immersed. After 1 h, a 1-ll spot of Pep 3ER1D5 peptide

was placed onto each slide in duplicate and allowed to

couple for 15min at 42 �C. The peptides were then

rinsed off and the slides were blocked in protein buffer.To determine the sensitivity to water immersion, the

amount of Pep 3ER1D5 peptide that coupled to the PI-

coated slides was measured by immunohistochemistry

(Materials and methods). Fig. 5A shows that the PI

groups� reactivity was unaffected by immersion in room

temperature water. Water is insufficiently nucleophilic to

displace the isocyanate protecting group at room tem-

perature. The reactivity of PI groups can be quenchedonly at higher temperatures, such as in boiling water

(data not shown).

Shelf life stability of PI slides

The shelf life stability of PI slides over time is also an

important practical consideration in facilitating the

manufacture of sensitive and reproducible assays.

de (0.5 pmol/spot) to PI slides, slides were either immersed (right bar) or

unaffected by immersion in water. The data represent the mean�SE of

he ability of PI slides to couple to either mouse IgG (12.5 ng) or the Pep

e mean and SE of triplicate measurements on the same slide are shown.


To address this question, we tested the stability of thePI-coated slides over time (Fig. 5B). On day 0, we coated

a large number of glass slides with the PI coating. These

slides were stored at 4 �C. During the month indicated

on the x axis (Fig. 5B), we applied 1 ll droplets (in

triplicate) of either Pep 3ER1D5 or mouse polyclonal

IgG. After 15min, the slides were rinsed and blocked.

The ability of the PI slide coating (after varying periods

of storage time) to couple the Pep 3ER1D5 or mouseIgG to fresh isocyanate-coated slides was measured by

immunohistochemistry, as described under Materials

and methods. These data illustrate that PI-coated slides

that were stored for 1 year have a coupling capability

comparable to that of freshly coated slides. The small

dip in the peptide (ER) group at 6–8 months is most

likely due to variability in the detecting assay procedure,

since the assay is performed by hand.

Peptides coupled to PI-coated slides have a long shelf life

In addition to the stability of the isocyanate chemis-

try, we tested the shelf life of the analytes after they are

coupled to PI glass slides. At the outset of this experi-

ment, we coupled replicate spots of the Pep 3ER1D5

peptide to PI slides. The peptide-coupled slides werestored at 4 �C. At various time points, as indicated on

the x axis of Fig. 6, the relative amount of the Pep

3ER1D5 peptide on the PI glass slide was tested using

the 1D5 mAb. The 1D5 mAb binds to the Pep 3ER1D5,

as previously described [3]. The presence of the 1D5

mAb was then detected using a standard labeled strep-

tavidin–biotin detection system (see Materials and

methods). Fig. 6 depicts the relative amount of detect-able ER peptide on the slides as a function of time in

storage. Fig. 6 shows that slides stored for 2 years prior

to testing had a comparable amount of Pep 3ER1D5

peptide relative to freshly prepared slides. These data,

Fig. 6. Peptide-coated PI slides are stable for up to 2 years. We coupled

0.5 pmol ER peptide to slides, which were stored at 4 �C until tested.

The relative amounts of immunoreactive Pep 3ER1D5 retained after

the indicated periods of time are represented as the spot intensity (y

axis) on a 1–256 scale. The average spot intensity is plotted with

standard errors bars.

therefore, indicate that peptides coupled onto PI slidesare stable for at least 2 years.

The PI coupling is covalent

Covalent bonds are highly durable and generally

more stable than noncovalent bonds. Based on our un-

derstanding of the chemical structure of the PI coupling

chemistry, we expected that analytes would bind cova-lently. However, we had not formally proven this point.

To assess the strength of the bond, we tested the ability

of a protein coupled to the PI-coated glass slide to

withstand elution under denaturing conditions. If ana-

lytes are covalently coupled to the PI glass slide coating,

then the analytes should not elute, even after treatment

with harsh denaturing buffers.

For this experiment, we used recombinant estrogenreceptor a (rER) that we coupled to the PI slide coating.

The 1D5 mAb binds to rER. We tested the ability of

rER to remain attached to the PI slide coating after

incubation in one of two different denaturing solutions,

each of which is designed to strip noncovalently bound

proteins off the surface. One of the treatments is to boil

the glass slides for 20min in 10mM citrate buffer, pH

6.0. The other is incubation in ECL buffer (see Materialsand methods) for 30min at 50 �C.

A flow chart of the experimental procedure is shown

in Fig. 7A. We first coupled varying amounts of rER to

the PI slide coating. The slides were then rinsed and

immersed in a protein blocking solution, to block un-

reacted PI groups. The rER-coated slides are subse-

quently incubated with the 1D5 ER mAb, which binds

to the rER on the glass slide. This is shown at the verytop of Fig. 7A and results in two proteins on the glass

slide. The first (rER) is presumably attached through a

covalent bond, whose strength we propose to test. The

second (1D5 ER mAb) is a noncovalent association. By

starting with both proteins, we can test both the dura-

bility of the PI bond and verify that the elution buffers

will, in fact, elute a noncovalently associated protein off

the glass slide.Positive control slides were not subjected to elution

treatment but were processed for immunohistochemical

detection of the 1D5 ER mAb. The positive control

thereby provides an indicator of the maximal colori-

metric intensity. Namely, the 1D5 ER mAb binds to the

rER on the PI-coated glass surface. The 1D5 ER mAb is

then detected with ‘‘detection reagents’’ (Fig. 7A),

comprising an anti-IgG–biotin conjugate followed bystreptavidin–HRP (Materials and methods). As Fig. 7B

illustrates, there is a progressive increase in color in-

tensity with increasing concentrations of rER on the

glass.

The experimental and negative control groups of

slides were then treated with either the citrate or the

ECL elution buffers. After treatment with one of the

Fig. 7. (A) Experimental flow chart to test whether the PI slide coating creates a covalent bond, such that analytes are covalently bound and cannot

be eluted off. (B) A protein coupled to a slide through the PI slide coating cannot be eluted off with denaturing buffers, supporting the concept that

the attachment bond is covalent. Varying amounts of rER analyte were initially bound to three groups of PI slides: positive control, experimental,

and negative control. The amount of rER incubated on the slide is indicated on the x axis. Two different elution conditions were tested for their

ability to elute off the rER. The data for citrate buffer elution are shown at left and ECL buffer elution data are shown at right. The relative amount of

rER analyte remaining on the slide is measured by the Spot Intensity (y axis) after immunohistochemical detection with the ER 1D5 mAb.



elution buffers, the negative control slide group was thenprocessed for immunohistochemical detection of the

1D5 mAb, if any still remained, on the glass slide (see

Materials and methods). Namely, the slide was incu-

bated with an anti-IgG–biotin conjugate, followed by an

incubation with streptavidin-HRP. Therefore, this neg-

ative control group tests whether the 1D5 mAb was in

fact eluted. Since antibodies bind to antigens through

noncovalent interactions, we would expect the elutiontreatment to remove the antibody. The negative control

is thereby a control on the elution process itself. If the

elution process worked properly, little to no colorimetric

signal should result in the negative control group. As

Fig. 7B demonstrates, virtually no color developed in

the negative control group, regardless of which elution

buffer was used.

The experimental group tests whether rER analyteelutes off the glass slide. It is shown toward the right side

of Fig. 7A. In the experimental group, the 1D5 mAb was

reapplied to the slides and incubated for 30min at RT.

The negative control demonstrated that the first appli-

cation of 1D5 did, in fact, elute. By reapplying a fresh

aliquot of the 1D5 ER mAb in the experimental group,

we can determine whether the rER also eluted off the

glass. Alternatively, if rER is still present on the glass,then the 1D5 mAb will bind to it. The 1D5 mAb was

then detected using detection reagents for mouse IgG

(Materials and methods). If the rER is covalently

bound, and therefore did not elute, then the color signal

intensity associated with the experimental group should

approximate the color signal intensity of the positive

control. On the other hand, if the rER is not covalently

bound, and therefore eluted off the glass, then the colorintensity associated with the experimental group should

approximate the color intensity of the negative control

Fig. 8. PI slides can immobilize carbohydrates to glass microscope slides. De

Varying concentrations of dextran, as indicated on the x axis, were coupled t

the slides was detected using one of three anti-dextran antibodies. Lipooligo

and WG, were serially diluted (fivefold) and coupled to isocyanate slides at th

glass was detected with anti-LOS mAb 2C7. The average color spot intensit

group. The data in Fig. 7B demonstrate that the color-imetric intensity of the experimental group approxi-

mated that of the positive control, indicating that the

analyte did not elute off. We conclude that the PI glass

slide coating creates a durable covalent bond.

PI slides covalently couple carbohydrates

Isocyanate groups can form carbamate bonds withmolecules containing hydroxyl groups, such as carbo-

hydrates. Therefore, we tested the capacity of PI slides

to immobilize carbohydrates to glass. We coupled either

dextran or lipooligosaccharide to PI-coated glass slides

at varying concentrations, as indicated on the x axis in

Fig. 8. The presence of the carbohydrate was detected

using mAbs to dextran or LOS. Three different mAbs to

dextran were used, as indicated in Fig. 8A. All threemAbs yielded generally similar results. Two different

types of LOS were tested for immobilization to PI slides.

The two are derived from N. gonnohorea strains 1523 or

WG, as indicated in Fig. 8B. Each of these also gave

similar results. The dextran or LOS mAbs were detected

using our standard labeled streptavidin–biotin IHC de-

tection protocol. We also found that the coupling ki-

netics and buffers for oligosaccharides were similar tothose of peptides or proteins (data not shown). These

data suggest that, like proteins and peptides, oligosac-

charides can also be readily coupled to glass through the

PI slide chemistry.

PI slides can also immobilize nucleic acids

Since most of our initial work was with peptidesand proteins, we were interested to determine whether the

PI slide coating chemistry can also attach DNA.

xtran (A) or lipooligosaccharide (B) were coupled to PI-coated slides.

o triplicate PI slides. The relative amount of dextran immobilized onto

saccharides (LOS), derived from two Nesseria gonnohorea strains 1523

e indicated concentrations on the x axis (B). LOS immobilized onto the

y of the peptide spots (y axis) is measured on a 1–256 scale.


Like carbohydrates, DNA also has hydroxyl groups towhich an isocyanate can bind, forming a carbamate bond.

This is in contrast to other covalent DNA attachment

chemistries, which require modification with an amine

prior to coupling. To test the PI slide for DNA arrays, we

immobilized purified PCR products derived from a

mouse cDNA library (see Materials and methods). The

clone set was printed in duplicate, without any modifi-

cation to the DNA onto PI-, aminosilane-, and poly-LL-lysine-coated slides. Non-DNA-binding areas were then

quenched by boiling the slide (as described in Materials

and methods). To probe the library, mouse brain mRNA

was extracted, reverse transcribed, and then labeled with

Cy5. The Cy5-labeled cDNA probe was hybridized to the

array overnight. A total of 2.5 lg of labeled cDNA was

hybridized. The slides were washed and scanned for Cy5

(excitation at 649 nm, emission at 670 nm). All the slidesare treated at the same time and under the same scan

conditionwithout any adjustment of images. Fig. 9 shows

Fig. 9. Demonstration of a DNA microarray on PI (center)-, aminosilane (lef

the area indicated in the dotted rectangles is shown at high magnification, bel

and methods) were printed in duplicate, without any modification to the D

mouse brain total RNA. The slides were scanned for Cy5.

the Cy5 emission images of the array. The images fromthe three slide coatings show a comparable or better

signal to noise ratio for PI as compared to aminosilane or

poly-LL-lysine slide coatings. Color intensity is represented

on a color scale, where white is the most and blue is the

least intense. The spot sizes and shapes are uniform,

indicating uniform DNA binding.

Discussion

We describe a new type of glass slide coating and its

performance characteristics. The coating is a modified

(protected) isocyanate group. To our knowledge, slide

coatings where the reactive group is an isocyanate are

not commercially available. Isocyanates tend to be

highly reactive, even with water. When an isocyanatereacts with water, a carbamic acid intermediate is

formed that ultimately generates an amine with the

t)-, and poly-LL-lysine (right)-coated glass slides. To show greater detail,

ow each image. PCR products of a mouse cDNA library (see Materials

NA. The library was probed with Cy5-labeled cDNA derived from a


release of CO2. The amine does not have the reactivitycharacteristics of an isocyanate, resulting in degradation

of the slide coating�s performance. Such instability can

be problematic during the printing of arrays. The hu-

midified environment of an array printer, although re-

quired to prevent evaporation of the array spots, could

result in degradation of the slides during the array

printing process. By contrast, the PI slide coating is

quite stable. It can be immersed in water for 1 h withoutdetectable degradation. The PI slide coating�s stability is

also reflected in the fact that we measured a shelf life of

at least 1 year when stored at 4–7 �C (Fig. 5).

We have previously described the chemical mecha-

nism of the PI slide coating�s stability [19]. Briefly, the

isocyanate is synthesized on the glass slide with a

blocking group. Water is not sufficiently nucleophilic to

displace the blocking group. Amines, hydroxyls, andcarboxyl groups, on the other hand, are sufficiently

nucleophilic to displace the blocking group and react

with the protected isocyanate. These characteristics re-

sult in a protected isocyanate with a threshold of reac-

tivity, below which the PI slide coating will not react.

This is, to our knowledge, the first description of a

stable isocyanate coupling chemistry for attachment of a

variety of different types of compounds to glass. Ourmethod of creating a PI slide coating entails a two-step

process. First, we add an aminosilane to the glass, re-

sulting in an abundance of amine groups protruding from

the glass surface. These amine groups are then converted

into protected isocyanate groups in a second incubation

step. An alternative method for creating isocyanate

groups on the glass surface includes adding a diisocya-

nate to the aminosilane coating. One arm of the diiso-cyanate attaches to the amine group, leaving the other

available for conjugation to a protein or peptide. In our

experience, this alternative approach (using phenylene-

diisocyanate) works, but yields lower-intensity signals,

suggesting inferior efficiencies of conjugation to proteins

and peptides (data not shown). The inferior performance

may be due to the possibility that both arms of the diis-

ocyanate become linked to adjacent amine groups on theaminosilane-coated glass. With both arms of the phen-

ylenediisocyanate occupied, it is incapable of attaching to

a protein or peptide. An alternative explanation for the

diisocyanate�s inferior performance characteristics is

that the phenylenediisocyanate slide became partially

quenched by water, since it does not have a protecting

group blocking reactivity with water.

We found that the PI slide coating displays a compa-rable sensitivity to previously described array coatings.

Array sensitivities have been characterized in two ways,

depending upon the purpose for which the array is used.

Some reports describe the lowest quantity ofmaterial that

can be attached to the substrate and still detected. Of

course, the amount of analyte bound to the glass slide will

also be directly affected by the size of the spot. In one

report that reviewed 11 different array surfaces [22], it wasfound that the sensitivity varied, depending upon the

antibody to be coated onto the substrate. In general, it

was found that the substrates allowed for reliable detec-

tion of 5 fmol per spot, which for IgG is 750 pg per spot

[22]. In that study, they applied spots of 1 nl or less. Ad-

justing for the volume of the spots applied to the glass, our

sensitivity appears to be in a comparable range. Namely,

our peptides were detectable at the nanogram per mi-croliter spot range, which is of a comparablemass/volume

ratio to the picogram per nanoliter spot level that was

previously described [22]. On a molar basis, our detection

sensitivity is in the femtomoles per microliter spot range.

Other reports describe the lowest concentration of

analyte in solution that can be detected using an array.

The detection sensitivities for analytes in solution have

been reported as 1–1600 ng/ml [23], 10 ng/ml [24], and15 ng/ml [25]. By employing particularly sensitive detec-

tion technologies, the sensitivity was further improved,

to the picogram per milliliter range [26–28]. We find

comparable sensitivity for the PI slide coating. Namely,

the ER 1D5 and the PR 636 monoclonal antibodies can

be detected at concentrations of 37 ng/ml (Fig. 3), the

lowest point on our scale and close to the limits reported

for conventional signal detection systems.While the sensitivity of the PI slide coating is com-

parable to that of other coatings, the PI slide has other

advantages. One of the attractive features of the PI slide

coating is its rapid reaction kinetics. At 40 �C, the re-

action was complete in 5min. This is superior to other

covalent coupling chemistries, such as epoxides or N -

hydroxysuccinimide esters, which often take hours or

overnight. Rapid coupling might be helpful in decreas-ing spot-to-spot variability due to evaporation. Shorter

coupling times mean that evaporation of the spot needs

to be prevented for only 5–15min. Premature evapora-

tion of a spot, before the moiety has coupled to the glass

surface, can result in abnormally low levels of coupling

and an artifactual negative result.

We demonstrated that PI slides are stable in water at

RT (Fig. 5A). The temperature of the water is importantin this regard, since we also found that boiling water will

both quench the protected isocyanate groups and largely

eliminate background fluorescence. For example, we

blocked the DNA array shown in Fig. 9 solely by boil-

ing. This sensitivity of the PI chemistry to boiling water

can be advantageous relative to other blocking solu-

tions. Blocking by just boiling in water could be helpful

where blocking with proteins may be a concern due to acompetitive displacement or masking of the coupled

molecules [29]. Blocking the PI slide by boiling is useful,

of course, only with analytes that will not be perma-

nently denatured by the treatment.

We found that the PI slides were highly stable during

storage at 4 �C. Figs. 4B and 5, both of which are time

course stability experiments, show some small degree of


fluctuation between time points. These fluctuationsprobably do not represent actual changes in stability.

Instead, it is most likely that this fluctuation is associ-

ated with a limitation in the experimental technique.

Namely, it is most likely due to the fact that each time

point in the graphs is actually a separate assay, run on

different days. These assays were processed by hand.

Consequently, slight variability in the assay procedure

will cause the observed fluctuation.Our data in Fig. 7 suggest that the printed PI slides

can be regenerated and reused. Such reusability of a

substrate, without compromising assay performance,

facilitates repeated use of the same substrate in multiple

assays using different probes. This feature effectively

decreases the cost of an assay. Repeated probing of the

same array with different probes also facilitates assay

comparisons. Reusable array substrates are an impor-tant consideration to some in choosing an array, par-

ticularly in the context of expression profiling [30].

In summary, with the stability problem addressed, the

PI slide coatingmay be particularly useful for the covalent

immobilization of small molecules, such as peptides

or oligonucleotides. Such small molecules often do not

attach well to poly-LL-lysine or other noncovalent coat-

ings. The PI coating would be an alternative, in thiscontext, to an epoxide or a succinimide ester slide coating.

We propose that the improved stability, rapid reaction

kinetics, strong bond, and versatility to couple a broader

range of biochemical compounds makes the PI slide

coating an attractive alternative.

Acknowledgments

This work was supported by Grants R44CA81950

and R43CA94557 from the National Cancer Institute to

S.A. Bogen.

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A water-stable protected isocyanate glass array substrate

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Transcript of A water-stable protected isocyanate glass array substrate