Acknowledgements
This work is supported by Guangdong Scientific and Technological Project
(2022A0505030024, 2022B1515120012), National Key Research and
Development Program of China (2022YFB3207200), National Natural Science
Foundation of China (62105221), Science and Technology Innovation
Commission of Shenzhen (JCYJ20220818102618040, JCYJ20200109105608771),
and Shenzhen Science and Technology Innovation Commission Sustainable
Development Project (KCXFZ20211020163814021).
Yiming Zhang, Zhi Chen and Songrui Wei contributed equally to this work.
Abstract: Real-time polymerase chain reaction (RT-PCR) remains
the most prevalent molecular detection technology for sewage analysis
but is plagued with numerous
disadvantages, such as time consumption, stringent equipment
requirements, and susceptibility to false negatives. In this study,
we construct an automated
robot-driven photoelectrochemical biosensing platform that utilizes the
CRISPR/Cas12a system,
to achieve fast, ultrasensitive,
high specificity detection of biological loads in sewage.
The
Shennong-1 robot integrates several functional modules, involving sewage
sampling and pretreatment to
streamline the sewage monitoring. A
screen-printed electrode is employed with a vertical graphene-based
working electrode and enhanced with surface-deposited Au nanoparticles
(NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the
double-stranded DNA anchored on Au nanoparticles. Using cDNA template of
Omicron BA.5 spike gene as a model, the photoelectrochemical biosensor
demonstrates excellent analytical performance, with a lower detection
limit of 2.93×102 zM and an outstanding selectivity at
the level of single-base mutation recognition.
Furthermore, the
rapid, accurate detection of BA.5
in sewage demonstrates the
feasibility of the photoelectrochemical platform for sewage
monitoring. In conclusion,
this platform allows early
detection and tracking of infectious disease outbreaks, providing timely
data support for public health institutions to take appropriate
prevention and control measures.
Keywords: robot automation, CRISPR/Cas12a system,
photoelectrochemical biosensor, nucleic acid detection, sewage
monitoring
1. Introduction
Sewage, a unique interface between
human beings and the natural environment, is generated by human
activities and responsible for a considerable biological load, primarily
from feces, saliva, and sputum. During the outbreak of the COVID-19
pandemic, numerous studies reported the presence of a large quantity of
COVID-19 virus in sewage[1-3]. In this context,
biological load, particularly with respect to microbial pathogens, has
attracted unprecedented attention[4,5]. On a
global scale, microbial pathogens present a substantial obstacle to
public health security and place a significant financial burden on the
healthcare system[6,7], particularly given that
three out of the top ten global health threats identified by the World
Health Organization in 2019 were directly linked to pathogens: pandemic
influenza, Ebola, and HIV[8].
Sewage is an essential medium for
capturing, accumulating, and transmitting
pathogens[9-11]; therefore, sewage has become an
ideal medium for monitoring the pathogens of the entire community, and
a powerful tool called
wastewater-based epidemiology has been developed to provide early
warning for the emergence of disease and track the outbreak
trend[5,12]. Various pathogens have been found in
sewage worldwide, including nearly all types of bacteria that can cause
human diseases, as well as viruses that have caused significant
outbreaks in recent epidemics and
pandemics[10,13,14]. Thus, it is extremely
essential to establish
a sewage monitoring platform for
surveillance of various pathogens.
RT-PCR is the most commonly
employed molecular detection technique in sewage analysis, alongside
other technologies utilized for this purpose[15].
RT-PCR demonstrates advantageous
qualities such as early diagnosis, high sensitivity, and high
specificity. However, this method has many disadvantages, including its
time-consuming nature, substantial technician requirements, the need for
laboratory cleanliness, testing equipment requirements, and
susceptibility to false negative results that are inconsistent with
clinical
manifestations[16,17]. In addition, traditional
sewage sampling, enrichment, concentration, nucleic acid extraction, and
amplification lack the support of automation technology, requiring an
investment of manpower and time, significantly reducing the timeliness
of sewage monitoring. Therefore, developing a rapid, highly specific,
and ultrasensitive surveillance platform for continuous monitoring of
biological loads in sewage is
necessary to obtain valuable information to effectively support disease
treatment, prevention, and control.
Recently, clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated proteins (Cas) technology has led to its
wide-spread recognition due to its potential application in molecular
biological diagnosis[18,19]. Compared with
traditional molecular diagnosis methods, the CRISPR/Cas nucleic acid
detection system has higher sensitivity, specificity, and
resolution[20]. Moreover, the CRISPR/Cas nucleic
acid detection system has a particular universality in biological
nucleic acid detection[21]. Different
CRISPR/Cas systems, including
DETECTR, HOLMES, and SHERLOCK, have been employed in the development of
various nucleic acid detection methods[20,22-25].
Recently, researchers have been incorporating CRISPR/Cas system with
various detection platforms to enhance the sensitivity of nucleic acid
detection methods[26-29]. Photoelectrochemical
(PEC) analysis has gained significant recognition as a versatile and
promising analytical technology in various
fields[30-32]. Because the light emission source
and electrical signal readout are completely separate, PEC sensors have
notable benefits including high sensitivity, minimal background
interference, rapid response time, and
affordability[33,34].
Therefore,
an
automated
robot-driven PEC biosensing platform
based on a CRISPR/Cas12a system was constructed to meet the need for
rapid, accurate detection of biological loads in sewage. The robot has
the capacity to automate the complete process, which includes sampling,
enrichment, concentration, nucleic acid extraction, and reverse
transcription. The electrode used
was a screen-printed electrode (SPE), and
the working electrode was based on
vertical graphene with
surface-deposited Au NPs. With the assistance of these Au NPs,
double-stranded DNA (dsDNA) was fixed
on the electrode surface.
Furthermore, CdTe/ZnS QDs were
fabricated on the electrode via an amide bond formed between the surface
carboxylic acid groups of CdTe/ZnS QDs and amino groups modified at the
5′ end of dsDNA, and the resulting electrode denoted as
CdTe/ZnS QDs–dsDNA/Au NPs/rGO
(reduced graphene oxide) electrode was obtained. When the Cas12a–crRNA
duplex was subjected to the target sequence, it specifically recognized
and combined with the target sequence, and trans-cleavage activity of
Cas12a was initiated. The dsDNA fixed on the electrode was
nonspecifically cut, resulting in CdTe/ZnS QDs detaching from the
electrode and reducing the photocurrent. The designed automated
robot-driven PEC biosensing platform showed excellent analytical
performance in the ultrasensitive detection of Omicron BA.5 and
potential application prospects in monitoring sewage.