A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis

Kwon, Soon-Young; Cho, Chang Hyun; Kwon, Youngmee; Lee, Eun Sook; Park, Je‐Kyun

doi:10.1038/srep45968

Cited by 22 publications

(16 citation statements)

References 39 publications

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“…For the IHC assay, we used a pressure-based reversible sealing method to seal the microfluidic channel by applying pressure on the chip that is in contact with the section slide. 19,24 However, the UV-curable PUA used in this work also has a rigid property after curing and causes a sealing problem. Since a typical microfluidic chip has a flat surface, it has a large contact area with the sample, thereby lowering the pressure applied to the channel surface.…”

Section: Resultsmentioning

confidence: 99%

“…19 An accurate IHC analysis for biomarker quantification was also demonstrated using a quantum-dot based microfluidic double-staining method, 20,21 a microfluidic tissue processor for formalin-fixed sections, 22 and frozen sections, 23 and standardization of immunostaining quality. 24 Although these microfluidic techniques have contributed to expand the potential of the IHC process, there are still a limited number of applications. This is due to the fact that most microfluidic IHC techniques are only applied to the immunostaining process of the entire IHC process, excluding the deparaffinization and antigen retrieval processes.…”

Section: Introductionmentioning

confidence: 99%

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Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section

et al. 2018

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We present here a novel microfluidic platform that can perform microfluidic on-chip immunohistochemistry (IHC) processes on a formalin-fixed paraffin-embedded section slide. Unlike previous microfluidic IHC studies, our microfluidic chip made of organic solvent-resistant polyurethane acrylate (PUA) is capable of conducting on-chip IHC processes consecutively. A narrow channel wall structure of the PUA chip shows effective sealing by pressure-based reversible assembly with a section slide. We performed both on-chip IHC and conventional IHC processes and compared the IHC results based on the immunostaining intensity. The result showed that the effects of the on-chip deparaffinization, antigen retrieval, and immunoreaction processes on the IHC result were equivalent to conventional methods while reducing the total process time to less than 1/2. The experiment with breast cancer tissue shows that human epidermal growth factor receptor 2 (HER2) classification can be performed by obtaining a clearly distinguishable immunostaining intensity according to the HER2 expression level. We expect our on-chip microfluidic platform to provide a facile technique suitable for miniaturized, automated, and precise diagnostic devices, including a point-of-care device.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section

et al. 2018

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“…Among the various microfluidic technologies, concentration gradient generation is one of the most useful technologies that can develop various concentration gradients at once, without the need for repetitive manual pipetting [1]. To date, microfluidic devices have been widely applied to generate the concentration gradient in various fields, such as cell-based drug screening [2,3], oil production screening from microalgae [4], immunohistochemistry [5], and antimicrobial susceptibility testing [6]. The different concentration of reagents is realized on a microscale, allowing rapid and high-throughput analysis compared to a bulk scale environment.…”

Section: Introductionmentioning

confidence: 99%

Finger-Actuated Microfluidic Concentration Gradient Generator Compatible with a Microplate

2019

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The generation of concentration gradients is an essential part of a wide range of laboratory settings. However, the task usually requires tedious and repetitive steps and it is difficult to generate concentration gradients at once. Here, we present a microfluidic device that easily generates a concentration gradient by means of push-button actuated pumping units. The device is designed to generate six concentrations with a linear gradient between two different sample solutions. The microfluidic concentration gradient generator we report here does not require external pumps because changes in the pressure of the fluidic channel induced by finger actuation generate a constant volume of fluid, and the design of the generator is compatible with the commonly used 96-well microplate. Generation of a concentration gradient by the finger-actuated microfluidic device was consistent with that of the manual pipetting method. In addition, the amount of fluid dispensed from each outlet was constant when the button was pressed, and the volume of fluid increased linearly with respect to the number of pushing times. Coefficient of variation (CV) was between 0.796% and 13.539%, and the error was between 0.111% and 19.147%. The design of the microfluidic network, as well as the amount of fluid dispensed from each outlet at a single finger actuation, can be adjusted to the user’s demand. To prove the applicability of the concentration gradient generator, an enzyme assay was performed using alkaline phosphatase (ALP) and para-nitrophenyl phosphate (pNPP). We generated a linear concentration gradient of the pNPP substrate, and the enzyme kinetics of ALP was studied by examining the initial reaction rate between ALP and pNPP. Then, a Hanes–Woolf plot of the various concentration of ALP was drawn and the Vmax and Km value were calculated.

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“…Microfluidic approaches have been employed for the efficient immunostaining by taking advantage of the high surface-to-volume ratio of microfluidic devices. [18][19][20][21] However, previous microfluidic systems are mainly targeting analysis of tissue samples and cannot be used for assaying suspended cells without individual cell trapping. Recently, hydrodynamic single-cell trapping methods have been developed for arraying suspended single cells and employed for the observation of cellular responses.…”

Section: Introductionmentioning

confidence: 99%

On-chip immunofluorescence analysis of single cervical cells using an electroactive microwell array with barrier for cervical screening

et al. 2019

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Several specific tests for cervical screening have been developed recently, including p16/Ki67 dual immunostaining for diagnosing high-risk human papillomavirus positive squamous intraepithelial lesion in the cervix. However, manual screening of cells in an entire glass slide is currently a standard clinical procedure for quantification and interpretation of immunocytochemical features of the cells. Here, we developed a microfluidic device containing an electroactive microwell array with barriers (EMAB) for highly efficient single-cell trapping followed by on-chip immunofluorescence analysis with minimum loss of the sample. EMAB utilizes patterned electrodes at the bottom of cell-sized microwells to trap single cells using dielectrophoresis (DEP) and cell-holding structures behind the microwells to stabilize the position of trapped cells even without DEP. Using the device, we evaluated the performance of p16/Ki67 dual immunostaining of HeLa cells on the chip. The device shows 98% cell-trapping efficiency as well as 92% cell-holding efficiency against the fixed HeLa cells, and we successfully demonstrated high-efficiency on-chip immunofluorescence analysis with minimal loss of sample. p16/Ki67 dual immunostaining using EMAB may be useful for complementary tests for cervical screening in confirming the histopathological diagnosis.

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A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis

Cited by 22 publications

References 39 publications

Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section

Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section

Finger-Actuated Microfluidic Concentration Gradient Generator Compatible with a Microplate

On-chip immunofluorescence analysis of single cervical cells using an electroactive microwell array with barrier for cervical screening

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