Real-time monitoring of cell viability by its nanoscale height change with oxygen as endogenous indicatorw

Scanning electrochemical microscopy (SECM) has been extensively applied in biological analysis such as measuring catalytic activity, monitoring the metabolism, investigating the neurotransmitter secretion, and imaging the morphology due to its ability in quantitative measurements of both intercellular and intracellular physiological processes. Unlike scanning ion conductance microscopy, these measurements usually need an exogenous redox indicator, such as Fe(CN)6 4 or Ru(NH3)6 3+ for morphological imaging, which makes it difficult to essentially exhibit the long-time state of cells due to the poor biocompatibility and toxicity of these indicators. Although atomic force microscopy (AFM) has been applied for high-resolution imaging of living cells, the physical interaction of AFMmay cause unexpected changes in cells. Thus, so far little attention has been paid to the long-time cell height change concerned with viability in a physiological environment. To address this issue, it is imperative to search for a biocompatible indicator for SECM analysis. Oxygen has been used as a redox probe for SECM study of the local catalytic activity of some biocatalytic systems. The formation and consumption of oxygen in cellular physiological processes have also been investigated by SECM. Since the oxygen reduction signal in SECM constant-height mode is usually composed of morphological effect and biological origin, this work designed a step-approaching strategy to distinguish the multiplex signals, and then used oxygen as an endogenous indicator for noninvasive SECM detection of cell height change. In order to obtain high spatial and temporal resolution for single-cell SECM analysis, this work firstly proposed a simple method for preparation of Pt nanodisk tips using a laser puller. The tips were prepared by assembling a Pt wire-inserted quartz capillary in a borosilicate glass tube for improving the ductibility (Fig. S1 in ESIw). Scanning electron microscopic images of the resulting Pt nanodisk tip showed an abrupt decrease of the tip diameter in the heating zone and an exposed Pt nanodisk (Fig. S2 in ESIw). The effective radius could be down to 5 nm calculated from the steady-state response of a redox probe. Considering that a small electrode had limited imaging range in constant-height mode, a 400-nm-radius tip was used in the work, and a potential of 0.4 V was applied for oxygen reduction (Fig. S3 in ESIw). Adherent BGC-823 human gastric carcinoma (BGC) cells were chosen as a model target for morphological study in phosphate-buffered saline (PBS). When the tip was laterally scanned in a horizontal (x–y) plane above a single BGC cell, the reduction current of oxygen was recorded as iT, which gave a negative peak iP at the center of the cell (Fig. 1a). A typical SECM image of a single BGC cell in PBS is shown in Fig. 1c. Although slight instability of the tip current was observed on the edge as reported by Bard’s group, currents around the cell were steady. The cell center taken by SECM was much darker than the edge, indicating the decrease of current, which apparently originated from three parts: (1) negative feedback effect due to cell morphology, (2) consumption due to cell respiration, (3) permeability for oxygen due to natural diffusion and SECM-induced transfer. Parts (2) and (3) could be involved in the net flux of oxygen through the cell membrane.