Black silicon as a platform for bacterial detection

Surface-enhanced Raman scattering (SERS) shows promise for identifying single bacteria, but the short range nature of the effect makes it most sensitive to the cell membrane, which provides limited information for species-level identification. Here, we show that a substrate based on black silicon can be used to impale bacteria on nanoscale SERS-active spikes, thereby producing spectra that convey information about the internal composition of the bacterial capsule. This approach holds great potential for the development of microfluidic devices for the removal and identification of single bacteria in important clinical diagnostics and environmental monitoring applications.Plasma etching of silicon can be used to produce inexpensive, large surface area, nano-textured surfaces known as black silicon. Recently, it has been shown that black silicon nano-needles can impale bacteria¹ and that it can be used as a sensor in microfluidic devices.² When coated by a plasmonic metal, such as gold, the nano-textured surface of black silicon is ideal for use as a surface-enhanced Raman scattering (SERS) sensing platform.³ This work aims to investigate whether gold-coated black silicon nano-needles can be used to both impale bacteria and identify them by SERS. This combination of properties would promote the development of microfluidic devices for the removal and monitoring of bacteria in a wide range of medical, environmental, and industrial applications.⁴Black silicon was fabricated by a reactive ion etching technique,⁵ resulting in pyramidal-shaped spikes of height 185 ± 30 nm, full width at half height of 54 ± 10 nm, and 10 ± 2.4 nm radius of curvature at the tip. Samples were then magnetron sputter coated with 200 nm of gold, as this coating thickness was found to provide a suitable compromise between SERS enhancement and impalement efficiency. E. coli (ATCC 25922) from −80 °C stock was isolated on a nutrient agar plate (Difco nutrient broth, Becton Dickinson) for approximately 12 h. A single E. coli colony was then inoculated from the plate into 20 ml of nutrient broth media and incubated overnight at 37 °C with orbital shaking at 200 rpm. The total biomass of overnight culture was adjusted to an optical density of 0.3 at λ = 600 nm by adding fresh sterile nutrient broth (Cary 50 spectrophotometer, Agilent). The E. coli planktonic cells were washed three times by centrifugation at 12 000 rpm (Centrifuge 5804 R, Eppendorf) for 2 min. The washed cells were then re-suspended in a low strength minimum medium (Dulbecco A, phosphate buffered saline). A volume of 100 μl of solution was pipetted onto substrates and left to incubate for 1 h on the bench. Separate sets of samples were created for scanning electron microscope (SEM) imaging, live/dead staining, and SERS. Three sets were needed as each of these measurements altered the samples and left them unsuitable for further analysis.The first set of samples was washed three times with milliQ water after incubation, allowed to dry and then immediately sputter coated with gold using the Emitech K975x (operating current 35 mA, sputter time 32 s, stage rotation 138 rpm, and vacuum of 1 × 10⁻² mbar). SEM imaging was performed with a Zeiss Supra 40VP in high vacuum mode with a working distance of 5 mm and an accelerating voltage of 3 kV. Figure ​Figure11 shows an example of the different levels of impalement that occurred on the black silicon surface. All cells showed signs of damage, but in some cases, the damage was limited to the perimeter of the cell and the main body appeared whole. In other cases, the entire cell had collapsed onto the spikes.Open in a separate windowFIG. 1.A typical SEM image showing E. coli cells with different levels of impalement on gold-coated black silicon.The second set of samples was used for live/dead staining (Invitrogen BacLight Bacterial Viability Kit L7012) with 3.34 mM SYTO 9 (green fluorescence) and 20 mM propidium iodide (red fluorescence). Equal volumes of both dyes were mixed thoroughly in a tube and added to the sample in a ratio of 3 μl of mixed dye to 1 ml of bacterial suspension. After mixing, a volume of 100 μl of the solution was pipetted onto the substrates, which were then incubated at room temperature in the dark for 15 min, before the staining solution was removed by pipetting. The substrates were then washed three times with milliQ water and mounted on a microscope slide for fluorescence imaging. The substrates were not allowed to dry and were stored in phosphate buffered saline at 4 °C when not in use. An epifluorescence microscope (Olympus IX71) with a mercury lamp source and a 60× water immersion objective was used to collect live/dead images from the substrates. Two filter blocks were used to collect the images: U-MNIBA2 blue excitation narrow band delivered green emission (live) and U-MWIG2 green excitation wide band provided red emission (dead).The live/dead image in Figure ​Figure22 shows a mix of both live and dead cells on the black silicon sample. The prevalence of live cells could be due to the incomplete impalement seen under SEM for some cells. It can also be explained by the sample still being wet during live/dead staining. The cells are dried prior to imaging in the SEM and this could weaken the cell wall and allow capillary forces to draw the cells onto the spikes for impalement. This hypothesis is supported by the large number of cells on the stained sample and the presence of cell groupings and cells imaged during mid-division. If the cells were immediately impaled, then such activity would not have been visible and a greater proportion of red cells would be expected.Open in a separate windowFIG. 2.Epifluorescence image showing live (green) and dead (red) E. coli cells after incubation on gold-coated black silicon.The third set of samples was washed three times with milliQ water after incubation and allowed to dry prior to spectral analysis. SERS spectra were collected with a Renishaw inVia Raman spectrometer operating at 785 nm with a 1200 l/mm grating. Power at the sample was 150 mW focused with a 100 × /0.85 NA objective to obtain a diffraction limited laser spot. The resulting spot size (≤2 μm in diameter) is well matched to the size of the bacterial cells. Spectra were collected with three accumulations of 10 s. Data were background subtracted⁶ and normalised to unity for ease of plotting. A great deal of variability was observed in the resulting spectra, as shown in Figure ​Figure33.Open in a separate windowFIG. 3.SERS spectra of E. coli after incubation on a gold-coated black silicon substrate. The spectrum numbers represent single cells at different locations and different levels of impalement.It should be noted that E. coli SERS is known to produce a high level of variability,^7–12 depending on the experimental setup.¹³ However, the variability seen in the SERS spectra of Fig. ​Fig.33 is unusual for measurements performed under consistent experimental conditions. This increased level of variability may be related to the different levels of impalement seen in Fig. ​Fig.1,1, which results in the probing of different internal components. SERS is a surface sensitive technique, with the signal primarily arising within 2 nm of the metal surface.¹⁴ Note that unlike apertureless nanoprobes¹⁵ or conical plasmonic nanotips,¹⁶ the SERS signal in black silicon arises primarily from “hot spots” between the spikes, where the plasmon resonance field is particularly strong.¹⁷ Therefore, depending on the depth and location of impalement, different biomolecules are expected to be excited by this novel substrate.Some peaks occur in the same position for multiple spectra (e.g., peak positions 420, 893, 1001, 1285, and 1307 cm⁻¹), but there are also a lot of unique peaks. The vertical lines in Fig. ​Fig.33 indicate peaks which have appeared in the literature for SERS of E. coli.^7–12 Spectrum 3 has a high proportion of peaks matching published values. This is also the case for spectrum 5, which shares a few peak positions with spectrum 3. Preliminary peak allocations have identified carbohydrates¹¹ (420 cm⁻¹), tyrosine¹¹ (650 cm⁻¹), adenine^10,11 (706 and 735 cm⁻¹), hypoxanthine⁷ (722 and1373 cm⁻¹), phenylalanine⁹ (1001 cm⁻¹), amide III (Ref. ¹⁰) (1285 cm⁻¹), CH₂ deformation¹² (1556 cm⁻¹), and C=C¹⁰ (1587 cm⁻¹).Given the varying levels of impalement observed in the SEM, it appears that the spike shape and Au coating should be further optimized to ensure that the entire cell is consistently pierced and the internal biomolecules are more comprehensively probed. In this way, it may be possible to obtain a more reproducible SERS spectrum of the internal biomolecular constituents of single bacterial cells, thereby providing rapid identification for medical and environmental diagnostic applications. Given that SERS is insensitive to water,⁴ future work should aim to achieve impalement in an aqueous environment, so that the full capability of microfluidics can be used to separate and concentrate suspended bacteria before presenting them to the substrate for rapid analysis.⁴ This suggests a broad range of potential applications in the detection, monitoring, and control of bacterial contamination.     

Understanding the lived experiences of novice out-of-field teachers in relation to school leadership practices

The lived experiences of novice teachers in out-of-field positions influence future career decisions and impact on their journey towards being competent and experienced practitioners, conversely their “life-world” is often misunderstood. The purpose of the study reported in this article is to investigate the lived experiences of these teachers, how principals’ understanding and leadership styles influence the lived experiences of novice out-of-field teachers, and what these lived experiences mean for school leaders. The article highlights perceptions of school leaders and novice out-of-field teachers about out-of-field teaching. It argues that the strategies implemented by school leaders based on their understanding of novice out-of-field teachers’ lived experience greatly influence the development of these teachers. It draws on Gadamer’s theories to investigate the lived experiences and perceptions of four principals and four novice out-of-field teachers through the different lenses of these participants. It concludes with a discussion on the interrelationships between school leaders’ understanding, novice teachers’ lived experience and what it means for the teaching environment. Participants’ interpretation of specific lived experiences connected to out-of-field teaching shapes meaning in their attempt to understand and to “belong,” for example, confidence issues, self-esteem concerns, and disconnectedness. The investigation of these units of meaning provides an in-depth understanding of the interrelationship between leadership and the lived experiences of novice out-of-field teachers.     

The effects of scientific representations on primary students’ development of scientific discourse and conceptual understandings during cooperative contemporary inquiry-science

Teaching students to use and interpret representations in science is critically important if they are to become scientifically literate and learn how to communicate their understandings and learning in science. This study involved 248 students (119 boys and 129 girls) from 26 grade 6 teachers’ classes in nine primary schools in Brisbane, Australia. Teachers were randomly allocated by school to one of three conditions: the contemporary science + representations condition (Experimental^a), the contemporary condition (Experimental^b), or the comparison condition as they participated in an eight-week inquiry-science unit on Natural Disasters. Data on students’ discourse were collected at two time points during the implementation of the unit and data on the concept maps were collected pre- and post-intervention while data on the reasoning and problem-solving (RP-S) task were collected following the intervention. The results show that when students participate in an inquiry-based science unit that is augmented with a variety of multimedia resources presenting a range of current contemporary events (Experimental^a and Experimental^b conditions), they demonstrate significantly more social language and basic scientific language and marked increases in moderate scientific language than their peers in the comparison condition. Interestingly, although there were no significant differences on the Personal Concept Map scores between the conditions at Times 1 and 2, the students’ scores in all conditions improved decidedly across time. It appears that as the children had more time to engage with the material, participate in cooperative peer discussions, and receive encouragement from their teachers to provide elaborated feedback to each other, their conceptual understandings of earthquakes were enhanced. However, although the children in the experimental conditions demonstrated significantly more social and scientific language than their peers in the comparison condition, these oral language skills did not transfer to the RP-S task, possibly because they may not have had enough time to consolidate their application in a novel context where they had to work independently.     

SMEC (her/his)tories

This collection of historical accounts provides diverse perspectives on the structure and culture of the community of researchers who completed their studies under the mentorship of researchers at SMEC. These her/histories provide multiple perspectives on the goals and values that motivated educators to become SMEC scholars.     

Understanding access to higher education amongst humanitarian migrants: an analysis of Australian longitudinal survey data

Higher Education - Humanitarian migrants are amongst the most marginalised population groups in countries within the Global North, including Australia. An important channel for these migrants to...     

Structural exclusion through school mathematics: using Bourdieu to understand mathematics as a social practice

Research on learner errors in mathematics education is beginning to focus on how teachers can learn to identify and engage with the reasoning behind these errors. Research on professional learning communities is beginning to show that they present powerful opportunities for on-going teacher collaboration and learning. In this paper, I bring the two areas of research together. Drawing on data from one professional learning community in the Data Informed Practice Improvement Project, I show how teachers in this community came to understand key concepts about learner errors and shifted their ways of talking about learner errors. I identify three important shifts that the teachers made in their learning about learner errors: from identifying to interpreting errors; from interpreting to engaging errors; and from focusing on learner errors to focusing on their own knowledge. I argue that these three shifts suggest a deepening of teachers’ thinking in relation to learner errors.     

The road to self-assessment: exemplar marking before peer review develops first-year students’ capacity to judge the quality of a scientific report

Lack of clarity about assessment criteria and standards is a source of anxiety for many first-year university students. The Developing Understanding of Assessment for Learning (DUAL) programme was designed as a staged approach to gradually familiarise students with expectations, and to provide opportunities for the development of the skills required to successfully complete assessment tasks. This paper investigated the students’ perceptions of the first two components of the DUAL programme, which assist first-year biology students to engage with stated assessment criteria and standards in order to develop their capacity to make judgements about scientific report exemplars, their peers’ scientific reports and ultimately their own. The study found strong evidence (96% of responses) that the marking and discussion of exemplar reports with peers and demonstrators clarified expectations of scientific report writing. A key feature of this element of DUAL was the opportunity for structured discussion about assessment criteria and standards between peers and markers (demonstrators). During these discussions, students can clarify explicit statements and develop a tacit knowledge base to enhance their ability to judge the quality of others’ work and their own. The peer review exercise (the second element of DUAL) was not rated as highly, with 65% of students finding the process helpful for improving their report. The negative reactions by a sizeable minority of students highlight the need to clearly communicate the expectations and benefits of peer review, with a focus on how the process of giving feedback to peers might benefit a student as much as receiving feedback on their own report.     

Correction to: What do upper‑elementary and middle school teachers know about the processes of text comprehension?

Reading and Writing -