Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins

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Abstract

A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 μm × 125 μm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

Original languageEnglish (US)
Pages (from-to)1080-1085
Number of pages6
JournalLab on a Chip - Miniaturisation for Chemistry and Biology
Volume6
Issue number8
DOIs
StatePublished - 2006

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Antibodies
Sepharose
Freezing
Melting point
Immobilized Enzymes
Proteins
Enzymes
Immunoglobulin G
Antibody Specificity
Plasma etching
Fluorescence microscopy
Enzyme activity
Photolithography
Photoresists
Antigens
Fluorescence Microscopy
Silicon Dioxide
Trypsin
Fluorescence
Silica

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins",
abstract = "A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 μm × 125 μm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70{\%} of immobilized enzyme activity.",
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AU - Lee, Lap Man

AU - Heimark, Ronald L

AU - Guzman, Roberto Z

AU - Baygents, James C

AU - Zohar, Yitshak

PY - 2006

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N2 - A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 μm × 125 μm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

AB - A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 μm × 125 μm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

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