Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers.

We have demonstrated a detection method for the ultra-sensitive detection of an mRNA biomarker. The method utilizes functionalized magnetic nanoparticles (MNPs) for signal enhancement in conjunction with surface plasmon resonance (SPR) on gold nanoslits. The approach for detection includes double hybridization at two different specific locations in two steps. First, the biomarker target molecule is captured with MNPs, and second, MNPs carrying the target molecule are introduced to the SPR chip to hybridize with probes immobilized on the gold nanoslits. In this work, MNPs were applied for a dual purpose: to isolate the target molecule from the sample matrix to prevent non-specific binding and to enhance the SPR response. Gold nanoslits that provide SPR sensing were fabricated by nanoimprinting lithography on polycarbonate (PC) film. The film was integrated with a microliter volume microfluidic chip to form the SPR detection chip. This detection method was used to detect mRNA heterogeneous nuclear ribonucleoproteins (hnRNP B1) in two cancer cell lines, CL1-0 and CL1-5. hnRNP B1 is an mRNA biomarker that is overexpressed in lung cancer tissue in the early stage of cancer and can be found in the serum and plasma of lung cancer patients. A synthetic target molecule and extracted total RNA from the cell lines were used as samples. Without amplification and labeling of the target molecule, the SPR results demonstrate a specific and sensitive method for the detection of hnRNP B1 mRNA in extracted RNA from the two selected cell lines. The method is capable of measuring down to 30 fM of the target molecule in a 7 µl sample (corresponding to 1.26 × 10(5) molecules) without amplification and labeling of the target molecule.

The Clinical Course of Early and Late Mild Cognitive Impairment.

Introduction: Amnestic mild cognitive impairment (MCI) can be classified as either early MCI (EMCI) or late MCI (LMCI) according to the severity of memory impairment. The aim of this study was to compare the prognosis and clinical course between EMCI and LMCI. Methods: Between January 2009 and December 2017, a total of 418 patients with MCI and 146 subjects with normal cognition were recruited from a memory clinic. All the patients received at least two series of neuropsychological evaluations each year and were categorized as either EMCI or LMCI according to Alzheimer's Disease Neuroimaging Initiative 2 (ADNI2) criteria. Results: In total, our study included 161 patients with EMCI, 258 with LMCI, and 146 subjects with normal cognition as controls (NCs). The mean follow-up duration was 3.55 ± 2.18 years (range: 1-9). In a first-year follow-up assessment, 54 cases (32.8%) of EMCI and 16 (5%) of LMCI showed a normal cognitive status. There was no significant difference between the first year EMCI reverter and NCs in terms of dementia-free survival and further cognitive decline. However, first-year LMCI reverters still had a higher risk of cognitive decline during the following evaluations. Until the last follow-up, annual dementia conversion rates were 1.74, 4.33, and 18.6% in the NC, EMCI, and LMCI groups, respectively. The EMCI and LMCI groups showed a higher rate of progression to dementia (log-rank test, p < 0.001) than normal subjects. Compared with NCs, patients in the LMCI group showed a significantly faster annual decline in global cognition [annual rate of change for the mini-mental status examination (MMSE) score: -1.035, p < 0.001]) and all cognitive domains, while those in the EMCI group showed a faster rate of decline in global cognitive function (annual rate of change for the MMSE score: -0.299, p = 0.001). Conclusion: It is important to arrange follow-up visits for patients with MCI, even in the EMCI stage. One-year short-term follow-up may provide clues about the progression of cognitive function and help to identify relatively low-risk EMCI subjects.

Altered mismatch response precedes gray matter atrophy in subjective cognitive decline.

The cross-sectional identification of subjective cognitive decline (SCD) in cognitively normal adults is particularly important for the early effective prevention or intervention of the future development of mild cognitive impairments (MCI) or Alzheimer's disease (AD). A pre-attentive neurophysiological signal that reflects the brain's ability to detect the changes of the environment is called mismatch negativity (MMN) or its magnetic counterpart (MMNm). It has been shown that patients with MCI or AD demonstrate reduced MMN/MMNm responses, while the exact profile of MMN/MMNm in SCD is substantially unknown. We applied magnetoencephalographic recordings to interrogate MMNm activities in healthy controls (HC, n = 29) and individuals with SCD (n = 26). Furthermore, we analyzed gray matter (GM) volumes in the MMNm-related regions through voxel-based morphometry and performed apolipoprotein E4 (APOE4) genotyping for all the participants. Our results showed that there were no significant differences in GM volume and proportions of APOE4 carriers between HC and SCD groups. However, individuals with SCD exhibited weakened z-corrected MMNm responses in the left inferior parietal lobule and right inferior frontal gyrus (IFG) as compared to HC. Based on the regions showing significant between-group differences, z-corrected MMNm amplitudes of the right IFG significantly correlated with the memory performance among the SCD participants. Our data suggest that neurophysiological changes of the brain, as indexed by MMNm, precede structural atrophy in the individuals with SCD compared to those without SCD.

In vitro electrical-stimulated wound-healing chip for studying electric field-assisted wound-healing process.

The wound-healing assay is an easy and economical way to quantify cell migration under diverse stimuli. Traditional assays such as scratch assays and barrier assays are widely and commonly used, but neither of them can represent the complicated condition when a wound occurs. It has been suggested that wound-healing is related to electric fields, which were found to regulate wound re-epithelialization. As a wound occurs, the disruption of epithelial barrier short-circuits the trans-epithelial potential and then a lateral endogenous electric field is created. This field has been proved invitro as an important cue for guiding the migration of fibroblasts, macrophages, and keratinocytes, a phenomenon termed electrotaxis or galvanotaxis. In this paper, we report a microfluidic electrical-stimulated wound-healing chip (ESWHC) integrating electric field with a modified barrier assay. This chip was used to study the migration of fibroblasts under different conditions such as serum, electric field, and wound-healing-promoting drugs. We successfully demonstrate the feasibility of ESWHC to effectively and quantitatively study cell migration during wound-healing process, and therefore this chip could be useful in drug discovery and drug safety tests.

Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.

In this paper, we report a new method to incorporate 3D scaffold with electrotaxis measurement in the microfluidic device. The electrotactic response of lung cancer cells in the 3D foam scaffolds which resemble the in vivo pulmonary alveoli may give more insight on cellular behaviors in vivo. The 3D scaffold consists of ordered arrays of uniform spherical pores in gelatin. We found that cell morphology in the 3D scaffold was different from that in 2D substrate. Next, we applied a direct current electric field (EF) of 338 mV/mm through the scaffold for the study of cells' migration within. We measured the migration directedness and speed of different lung cancer cell lines, CL1-0, CL1-5, and A549, and compared with those examined in 2D gelatin-coated and bare substrates. The migration direction is the same for all conditions but there are clear differences in cell morphology, directedness, and migration speed under EF. Our results demonstrate cell migration under EF is different in 2D and 3D environments and possibly due to different cell morphology and/or substrate stiffness.

Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.

We report a new design of microfluidic chip (Multiple electric Field with Uniform Flow chip, MFUF chip) to create multiple electric field strengths (EFSs) while providing a uniform flow field simultaneously. MFUF chip was fabricated from poly-methyl methacrylates (PMMA) substrates by using CO2 laser micromachining. A microfluidic network with interconnecting segments was utilized to de-couple the flow field and the electric field (EF). Using our special design, different EFSs were obtained in channel segments that had an identical cross-section and therefore a uniform flow field. Four electric fields with EFS ratio of 7.9:2.8:1:0 were obtained with flow velocity variation of only 7.8% CV (coefficient of variation). Possible biological effect of shear force can therefore be avoided. Cell behavior under three EFSs and the control condition, where there is no EF, was observed in a single experiment. We validated MFUF chip performance using lung adenocarcinoma cell lines and then used the chip to study the electrotaxis of HSC-3, an oral squamous cell carcinoma cell line. The MFUF chip has high throughput capability for studying the EF-induced cell behavior under various EFSs, including the control condition (EFS = 0).