Single-drop analysis of various proteases in a cancer cell lysate using a capillary-assembled microchip

Abstract

Single-drop analysis of two different real sample solutions (2 microL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5-8.2 (% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery.

In vitro anticancer activity of acetylshikonin action against cervical cancer

“…Cervical cancer is currently ranked as the second most common cause of cancer-related morbidity and the third most common cause of mortality worldwide (O'Keefe et al, 2009). Treatment of cervical cancer has typically been viewed as surgical with possible adjuvant therapy versus initial radiotherapy and platinum-based chemotherapy (Yue et al, 2008;Henares, Mizutani, Sekizawa & Hisamoto, 2008;Kelly et al, 2010). But the combination treatment has significant risk of neutropenia, anemia, and thrombocytopenia side effects, also result in drug resistance.…”

Fabrication and packaging of a mass-producible capillary-assembled microchip for simple and multiplexed bioassay

“…Thus, simple and multiplexed enzyme activity assay was successfully demonstrated. Since we have already shown the similar multiplexed enzyme activity assay using cell lysate sample to evaluate anticancer drug [31], present device is also expected to be applied for the real sample analysis.…”

“…Furthermore, since the present CAs-CHIP-3G does not require complicated operations and bulky pump system, implementation in point-of-care applications is also expected. Furthermore, CAs-CHIP-3G allowed high success rate of close to 100% for sample introduction compared with CAs-CHIP-1G [23,30,31], and from the view point of mass production, CAs-CHIP-3G is clearly advantageous compared with 1G and 2G [35], which require one-by-one embedding of capillaries into PDMS channels.…”

“…We have developed various glass capillary biosensors to be integrated into CAs-CHIP, to measure various analytes such as proteins [25][26][27][28], ions [29,30], enzymes [30,31], and metabolites [32,33] Among many capillary biosensors developed, dry reagentrelease capillary (dRRC) which allows spontaneous dissolution of analytical reagents and selective reaction to give fluorescence response by sample introduction via capillary action, played an important role for the simplicity in capillary biosensor preparation and biosensing [30]. However, both the first (CAs-CHIP-1G) [23,30,31] and second (CAs-CHIIP-2G) [35] generations of CAs-CHIP requires the meticulous one-by-one capillary embedding onto a PDMS microchannel. This issue could limit its ease of fabrication, usability, and diminish the potential for mass production.…”

Highly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates

“…[12][13][14][15][16][17][18][19][20][21][22][23][24][25] Especially, for enzyme activity assays, 18-23 the inner surface of a capillary is covered with a soluble coating containing reagents for the enzyme activity assay. Introducing a sample solution containing enzymes into the RRCs by a capillary action allows for the spontaneous release of the reagents and subsequent mixing with the solution.…”