A high stable sample loading for analysis of adult alpha-thalassemia via the improved microarray isoelectric focusing of Hb species.

The isoelectric focusing has realized various improvements, including the protocols and creation of mIEF (microcolumn isoelectric focusing) instruments with excellent sensitivity for screening of diabetes and beta thalassemia. However, the problem of manual sample loading and hydration for the mIEF limits the operational capacity for stably detecting and quantitating most abnormal hemoglobin (Hb). Herein, we provided a high stable sample loading protocol for analysis of alpha thalassemia and Hb variants. In contrast to the previous volume of 20 µl, a 100 µl blood sample solution in this protocol was optimized with mixture of 6.4-7.5 and 3-10 pH carrier ampholytes, pI markers and loaded for 30 mins IPG microcolumn hydration. The hydrated microcolumn was then automatically loaded onto the mIEF chip array to which CH3COOH and NH4OH act as anodic and cathodic solutions. Lastly, the IEF was run for 9 mins. Hb H, Barts, A1c, F, A2 and CS were simultaneously separated and focused with higher resolution and sensitivity in quantifying H and Barts as low as 0.6 and 0.5 % respectively. Accordingly, there was an enhanced stability and linearity with a rapid assay time of 45 secs per sample. Moreover, analysis showed a fitting linear relationship with conventional technology at R2 = 0.9803 for H and R2 = 0.9728 for Barts thereby indicating greater accuracy confirmed by the AUC. Hence, the developed protocol could simply be employed for high stable and throughput batch sample loading of hydration, and accurate separation and quantitation of Hb variants for alpha and beta thalassemia.

A temperature-independent model of dual calibration standards for onsite and point-of-care quantification analyses via electrophoresis titration chip.

Electrophoresis titration chip (ETC) is a versatile tool for onsite and point-of-care quantification analyses because it affords naked-eye detection and a straightforward quantification format. However, it is vulnerable to changes in environmental temperature, which regulates the electrophoretic migration by affecting the ion mobility and the target recognition by influencing the enzyme activity. Therefore, the quantification accuracy of the ETC tests was severely compromised. Rather than using the dry bath or heating/cooling units, we proposed a facile model of dual calibration standards (DCS) to mathematically eliminate the effects of temperature on quantification accuracy. To verify our model, we deployed the ETC device at different temperatures ranging from 5 to 40 °C. We further utilized the DCS-ETC to determine the protein content and uric acid concentration in real samples outside the laboratory. All the experimental results showed that our model significantly stabilized the quantification recovery from 35.31-153.44 % to 99.38-103.44 % for protein titration; the recovery of uric acid titration is also stable at 96.25-106.42 %, suggesting the enhanced robustness of the ETC tests. Therefore, DCS-ETC is a field-deployable test that can offer reliable quantification performance without extra equipment for temperature control. We envision that it is promising to be used for onsite applications, including food safety control and disease diagnostics.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.